Tài liệu Báo cáo khoa học: Open questions in ferredoxin-NADP+ reductase catalytic mechanism ppt

REVIEW ARTICLE
Open questions in ferredoxin-NADP
+
reductase catalytic mechanism
Ne
´
stor Carrillo and Eduardo A. Ceccarelli
Molecular Biology Division, Instituto de Biologı
´
a Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioquı
´
micas y
Farmace
´
uticas, Universidad Nacional de Rosario, Argentina
Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are
ubiquitous flavoenzymes that deliver NADPH or low
potential one-electron donors (ferredoxin, flavodoxin) to
redox-based metabolisms in plastids, mitochondria and
bacteria. The plant-type reductase is also the basic prototype
for one of the major families of flavin-containing electron
transferases that display common functional and structural
properties. Many aspects of FNR biochemistry have been
extensively characterized in recent years using a combination
of site-directed mutagenesis, steady-state and transient kine-
tic experiments, spectroscopy and X-ray crystallography.
Despite these considerable advances, various key features in
the enzymology of these important reductases remain yet to
be explained in molecular terms. This article reviews the
current status of these open questions. Measurements of
electron transfer rates and binding equilibria indicate that

NADP(H) and ferredoxin interactions with FNR result in a
reciprocal decrease of affinity, and that this induced-fit step is
a mandatory requisite for catalytic turnover. However, the
expected conformational movements are not apparent in the
reported atomic structures of these flavoenzymes in the free
state or in complex with their substrates. The overall reaction
catalysed by FNR is freely reversible, but the pathways
leading to NADP
+
or ferredoxin reduction proceed through
entirely different kinetic mechanisms. Also, the reductases
isolated from various sources undergo inactivating dena-
turation on exposure to NADPH and other electron donors
that reduce the FAD prosthetic group, a phenomenon that
might have profound consequences for FNR function
in vivo. The mechanisms underlying this reductive inhibition
are so far unknown. Finally, we provide here a rationale to
interpret FNR evolution in terms of catalytic efficiency.
Using the formalism of the Albery–Knowles theory, we
identified which parameter(s) have to be modified to make
these reductases even more proficient under a variety of
conditions, natural or artificial. Flavoenzymes with FNR
activity catalyse a number of reactions with potential
importance for biotechnological processes, so that modifi-
cation of their catalytic competence is relevant on both
scientific and technical grounds.
Keywords: ferredoxin-NADP(H) reductase; flavoproteins;
oxidoreductases; ferredoxin; flavodoxin; catalytic
mechanism; X-ray crystallography; steady-state kinetics;
transient kinetics; enzyme evolution.

Portrait of a reductase
Ferredoxin-NADP(H) reductases (FNR, EC 1.18.1.2) con-
stitute a family of hydrophilic, monomeric enzymes that
contain noncovalently bound FAD as a prosthetic group.
The first FNR was isolated from pea thylakoids in the mid-
1950s [1]. Shortly thereafter, Shin and Arnon [2] showed
that the physiological role of the chloroplast reductase was
to catalyse the final step of photosynthetic electron trans-
port, namely, the electron transfer from the iron-sulphur
protein ferredoxin (Fd), reduced by photosystem I, to
NADP
+
(Eqn 1). This reaction provides the NADPH
necessary for CO
2
assimilation in plants and cyanobacteria.
2FdðFe
2þ
ÞþNADP
þ
þ H
þ
!

2FdðFe
3þ
Þ
þ NADPH ð1Þ
Equation 1 reflects one of the most conspicuous features of
FNR, its ability to split electrons between obligatory one-

and two-electron carriers, which is a direct consequence of
the biochemical properties of its prosthetic group. FAD and
other flavins (Fl) can exist in three different redox states:
oxidized, one-electron reduced (semiquinone) radical and
fully reduced hydroquinone, containing, respectively, 18, 19
and 20 electrons in a p orbital system constructed from 16p
atomic orbitals [3]. The isoalloxazine ring also has 7r lone
electron pairs available for protonation, providing ample
opportunity for tautomers.
When free in solution, flavin semiquinone radicals
disproportionate to the oxidized and reduced forms
(Eqn 2), but when buried in a protein they are much less
prone to dismutation. This results in a considerable
stabilization of the semiquinone, which in turn allows
flavoproteins, in general, to readily engage in mono- and
Correspondence to N. Carrillo, IBR, Facultad de Ciencias
Bioquı
´
micas y Farmace
´
uticas, Universidad Nacional de Rosario,
Suipacha 531 (S2002LRK) Rosario, Argentina.
Fax: + 54 341 439 0465, Tel.: + 54 341 435 0661,
E-mail:
Abbreviations:cytc,cytochromec; Fl, flavin; Fd, ferredoxin;
FNR, ferredoxin-NADP(H) reductase; Fld, flavodoxin;
ox, oxidized; red, reduced; sq, semiquinone.
Enzyme: Ferredoxin-NADP(H) reductases (FNR, EC 1.18.1.2).
(Received 13 January 2003, revised 4 March 2003,
accepted 13 March 2003)

Eur. J. Biochem. 270, 1900–1915 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03566.x
bielectronic exchange reactions. This disposition is favoured
in FNR by the small differences existing in the redox
potentials (E
m
) for the various one- and two-electron
transfer processes, that is )331, )314 and )323 mV for
the oxidized/semiquinone couple, the semiquinone/hydro-
quinone pair and the two-electron FAD reduction, respect-
ively.
H
2
Fl
Æ
þ H
2
Fl
Æ
!

HFl
ox
þ H
2
Fl
À
red
þ H
þ
ð2Þ

FNR functions are by no means confined to photosyn-
thesis. Following the isolation of the chloroplast reductase,
flavoproteins with FNR activity were found in photo-
trophic and heterotrophic bacteria, animal and yeast
mitochondria, and apicoplasts of obligate intracellular
parasites [4–10]. Studies in a number of species showed
that FNR operates as a general electronic switch at the
bifurcation steps of many different electron transfer
pathways. In heterotrophic organisms and tissues, inclu-
ding roots and other nonphotosynthetic plant organs, the
reaction represented by Eqn (1) is driven toward Fd
reduction, mobilizing low-potential electrons for a diversity
of metabolisms. They include steroid hydroxylation, nitrate
reduction, nitrogen and hydrogen fixation, anaerobic
pyruvate assimilation, desaturation of fatty acids and
synthesis of amino acids and deoxyribonucleotides (Table 1,
reviewed in [11]). Van Thor et al. [12] have proposed that
this ÔbackwardÕ reaction might also occur in photosynthetic
cells of cyanobacteria, being a rate-limiting step of cyclic
electron flow around photosystem I.
Some bacteria and algae possess another flavoprotein,
FMN-containing flavodoxin (Fld), that is able to effi-
ciently replace Fd as the electron partner of FNR in
different metabolic routes, including photosynthesis [8].
In cyanobacteria, Fld expression is induced under condi-
tions of iron deficit, when the [2Fe)2S] cluster of Fd
cannot be assembled [8]. In other prokaryotes, flavodoxins
are synthesized constitutively, or induced by oxidants
[13]. Both Fld and FNR participate in detoxification of
reactive oxygen species in aerobic and facultative bacteria

[13,14].
FNR displays a strong preference for NADP(H) and is a
very poor NAD(H) oxidoreductase. In contrast, the site of
electron donation from reduced flavin appears to be open to
a remarkable variety of adventitious oxidants of very
different structure and properties. The reaction is largely
irreversible and led the original FNR discoverers to label it
as a thylakoid-bound ÔNADPH diaphoraseÕ [1].
NADPH þ H
þ
þ nA
ox
! NADP
þ
þ nA
red
ð3Þ
A
ox
and A
red
represent the oxidized and reduced forms of
the electron partner, and the term n equals one or two
depending on whether the oxidant behaves as a two- or a
one-electron carrier, respectively. The list of acceptors
comprises ferricyanide and other complexed transition
metals, substituted phenols, nitroderivatives, tetrazolium
salts, NAD
+
(transhydrogenase activity), viologens, qui-

nones and cytochromes (reviewed in [15]). By contrast, the
oxidase activity of FNR is very low [15,16], suggesting that
there might be restrictions to the formation of the caged
radical pair and/or the covalent (C4a)-flavin hydroperoxide
intermediates required for efficient oxygen reduction [17],
although other mechanisms cannot be ruled out. The
oxidase reaction is enhanced several-fold by many FNR
acceptors, including one-electron reduced Fd or Fld,
viologens, nitroderivatives and quinones, that can readily
engage in oxygen-dependent redox cycling leading to
superoxide formation [16,18]. Diaphorase activity is prob-
ably devoid of physiological meaning in most cases, but it
has paid an enormous service to the understanding of FNR
function and catalytic mechanism. In addition, some of
these artificial reactions might have technological relevance
for bioremediation and the pharmaceutical industry [18,19].
This brief account illustrates the plasticity of FNR as a
catalyst and its ubiquity among living organisms. Studies on
the enzymology of this reductase began in the late 1960s,
employing both steady-state and rapid kinetic measure-
ments, and a comprehensive model describing the various
steps of reaction 1 was formulated by Batie and Kamin in
1984. Since then, an impressive amount of information on
FNR structure, function and biogenesis has been gathered,
providing clues to understand holoenzyme assembly, sub-
strate binding and catalysis in molecular terms. However,
several key features of FNR enzymology remain obscure,
and could not be accounted for, or reconciled with the
available structural-functional data. The aim of this article is
to review these open questions in FNR biochemistry, as a

tool and conceptual framework for future research. It is
worth noting that a thorough understanding of FNR
catalytic mechanism has become increasingly important
after the recognition of this reductase as the prototype for a
Table 1. Functions associated with ferredoxin-NADP(H) reductase in different organisms.
Organism or tissue Function Related partner or metabolism References
Chloroplasts NADP
+
reduction Photosynthetic electron transport [2]
cyanobacterial vegetative cells [8]
Nonphotosynthetic plastids Fd reduction Nitrogen assimilation [72,73]
Cyanobacterial heterocysts Fd or Fld reduction Dinitrogen fixation [8]
Mitochondria Fd reduction Steroid hydroxilation [58]
Fatty acid desaturation
E. coli Fld reduction Activation of anaerobic enzymes [7]
E. coli NADPH oxidation Protection against oxidative stress [14]
Clostridia Fd reduction Hydrogen fixation [4]
Methanogenic bacteria Fd reduction Methane assimilation [5]
Apicomplexan parasites Fd reduction Fatty acid desaturation [10]
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1901
large family of flavin-containing enzymes displaying similar
structures and reaction mechanisms [20–26].
The two-domain structure of ferredoxin-
NADP(H) reductases
Three-dimensional models of the oxidized and fully reduced
forms of spinach FNR, refined to 1.7 A
˚
resolution, were
first reported by Karplus and coworkers [20–23]. The
flavoprotein molecule is made up of two structural domains,

each containing approximately 150 amino acids (Fig. 1A).
The carboxyl terminal region includes most of the residues
involved in NADP(H) binding, whereas the large cleft
between the two domains accommodates the FAD group.
The isoalloxazine ring system is tightly bound to the amino
terminal domain through hydrogen bonds and van der
Waals contacts [23]. It stacks between the aromatic side
chains of two tyrosine residues, represented in spinach FNR
by Tyr96 on the si-face and Tyr314 (the carboxyl terminus)
on the re-face (Fig. 1B). The phenol ring of Tyr314 and the
flavin group are coplanar in such a way as to maximize
p-orbital overlap [20]. A large portion of the isoalloxazine
moiety is shielded from the bulk solution by the side chain
of the carboxyl terminal tyrosine, but the edge of the
dimethyl benzyl ring that participates in electron transfer
remains exposed to solvent in the native holoenzyme [23].
Crystal structures have also been resolved for other FNR
proteins, including those present in pea, paprika and maize
chloroplasts [27–29], corn root plastids [30], cyanobacteria
[31], Escherichia coli [32] and Azotobacter vinelandii [33].
Despite ample variations in amino acid sequences, the chain
topologies of all these proteins are highly conserved, with
most differences occurring at the loops between the
invariant secondary structure elements [20–23,27–33]. In
the chloroplastic and cyanobacterial reductases, as in most
flavoproteins, the FAD molecule binds in an extended form,
with the 2¢-P-AMP moiety wrapped up by a short sheet-
loop-sheet motif of the apoprotein [20–23,27–31]. In
contrast, the adenosine in the E. coli FAD bends back
from the diphosphate so that the nitrogen at position 7 of

the adenine group forms a hydrogen bond to nitrogen 1 of
the isoalloxazine. This interaction is further stabilized by
stacking of the two terminal aromatic side chains; the
phenol ring of Tyr247 with the flavin, as in plant FNR, and
the indole of Trp248 with the adenine [32].
The FAD cofactor of the A. vinelandii FNR is also twisted
and stabilized in a similar way, but the environment of the
prosthetic group presents some unique features in this
reductase. The most important difference is the absence of
the aromatic interaction on the re-face of the isoalloxazine
that is typical of plant, cyanobacterial and E. coli FNR
proteins [33]. Instead, the tyrosine position in the Azotobacter
enzyme is occupied by an alanine (Ala254). Despite these
major modifications in the active site region, the FMN halves
of FAD display very similar conformations when the various
homologous proteins are superimposed [20–23,27–33].
Several amino acid residues important for the structural
integrity of the native holoenzyme, for electron transfer, and
for FAD, NADP(H), ferredoxin and flavodoxin binding,
have been identified using a combination of chemical
modification experiments, site-directed mutagenesis and
X-ray diffraction studies. This very interesting aspect of
FNR biochemistry will only be addressed here in relation to
the catalytic mechanism of the enzyme. Further information
on these topics can be found in a number of articles and
reviews [23,31,34–57].
Finally, it is noteworthy that not all flavoenzymes
displaying FNR activity belong to the FNR class. The
adrenodoxin reductases found in animal and yeast mito-
chondria and their bacterial homologues represent a curious

case. They are hydrophilic, monomeric proteins made up of
FAD and NADP(H) domains that can freely exchange
electron partners (ferredoxin, flavodoxin, adrenodoxin)
with plant-type FNR [58]. Many features of NADP(H)
docking and catalysis are also similar, although reaction
geometry is different [58]. However, mitochondrial reduc-
tases are unrelated in sequence to their chloroplast coun-
terparts, and the structural data indicate that they actually
belong to the disulphide oxidoreductase family of flavopro-
teins, whose prototype is glutathione reductase [6,58]. The
plant-type and mitochondrial-type FNR progenies thus
represent two different and independent origins, followed by
a remarkable case of convergent evolution to yield proteins
with essentially the same enzymatic properties.
The reaction pathway
Batie and Kamin [59,60] formulated the first detailed
pathway for the FNR-mediated electron transfer between
NADP(H) and ferredoxin, using data from binding equili-
bria, steady-state kinetics and rapid mixing experiments
(Fig. 2). The overall reaction was interpreted as an ordered
two-substrate process, with NADP
+
binding first. Under
these assumptions, the kinetics were shown to be consistent
with the formation of ternary complexes as intermediates of
the catalytic mechanism (Fig. 2, steps 2 and 5). Substrate-
binding parameters and rate constants were determined for
the complete pathway mediated by both plant and bacterial
reductases, and for several individual steps (Table 2). Turn-
over numbers in the range of 200–600 s

)1
have been reported
for the spinach and Anabaena enzymes, whereas E. coli
FNRismuchlessactive(Table2).Go
´
mez-Moreno and
coworkers have also studied the electron transfer to and from
flavodoxin [46,47 and references therein]. The reverse
reaction, that is the electron transport from NADPH to Fd
(or Fld), is routinely measured in vitro through a coupled
assay, using cytochrome c (cyt c) as final electron acceptor
(Eqns 4 and 5).
NADPH þ 2FdðFe
3þ
Þ
!

NADP
þ
þ H
þ
þ 2FdðFe
2þ
Þð4Þ
Fd ðFe
2þ
Þþcyt cðFe
3þ
Þ!FdðFe
3þ

Þþcyt cðFe
2þ
Þð5Þ
In the following sections, we will analyse the various steps
involved in FNR catalysis, with emphasis in unresolved or
controversial questions. Most of the discussion will be based
on data obtained with the plant and cyanobacterial
flavoenzymes. The reductases present in E. coli and other
bacterial species are less well characterized, but it is already
clear that they display a number of differences with respect
to their plant-type counterparts. These distinct features will
also be addressed when pertinent.
1902 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 1. The Ca polypeptide backbone and the active site region of plant-type ferredoxin–NADP(H) reductase. (A) FNR is a two-domain flavoprotein.
The computer graphic is based on X-ray diffraction data for the spinach enzyme [23], with the FAD binding domain shown in blue, the NADP(H)
binding domain in pink, and the FAD prosthetic group in yellow. (B) Detailed view of the isoalloxazine ring system in FNR, displaying relevant
interactions with active site amino acid residues. The phenol ring of the carboxyl terminal tyrosine 314 shields the re-side of the flavin from solvent.
The figure was drawn using
SWISS
-
PDBVIEWER
3.7 and rendered with
POV
-
RAY
.
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1903
NADP(H) binding (Fig. 2, steps 1 and 9)
Even though no chemistry is expected to occur between the
oxidized nicotinamide and the oxidized flavin, the role of

NADP
+
as leading substrate during FNR turnover is
supported by a number of kinetic measurements. In the
spinach enzyme, electron transfer from reduced Fd to FNR
is too slow (k
obs
¼ 40–80 s
)1
) to account for steady-state
rates of nucleotide reduction (k
obs
>500s
)1
). NADP
+
binding greatly accelerates this reaction (Table 2), indicating
that the presence of the coenzyme at the active site is a
prerequisite for catalysis [60]. Possible mechanisms involved
in this activation are discussed in the next section.
Formation of the binary complex has been measured
in vitro by a variety of techniques, most conspicuously
differential spectroscopy. Binding isotherms fit to simple
hyperbolic functions in all cases. Dissociation constants
increased with the ionic strength of the medium (I)andthe
Fd concentration, with K
d
¼ 6–20 l
M
at I % 100 m

M
for
plant and Anabaena reductases [43,44,50,59,61]. Karplus
and coworkers [20,23] have proposed that stacking of the
nicotinamide ring onto the re-face of the isoalloxazine
moiety during enzyme turnover requires displacement of the
aromatic side chain of the carboxyl terminal tyrosine. This
thermodynamically unfavoured process results in a decrease
of the binding affinity for NADP(H) relative to those of the
2¢-P-AMP and 2¢-P-ADP-ribose analogues [61]. A similar
movement has been postulated for the penultimate tyrosine
residue of E. coli FNR, although in this case the carboxyl
terminal tryptophan interacting with the adenosine moiety
of FAD might also undergo a conformational change upon
substrate binding [32,62–64].
Complex formation is then interpreted as a two-step
binding of the nucleotide to a bipartite site (Fig. 3). The first
step involves a strong interaction of FNR with the
adenosine part of NADP(H), followed by isomerization
leading to nicotinamide docking and, eventually, hydride
transfer (Eqn 6).
FNR þ NADPðHÞ
!

FNR NADPðHÞ
!

FNR NADPðHÞð6Þ
Where FNRÆNADP(H) and FNRÆNADP(H) represent
complexes with the coenzyme bound through the adeno-

sine, or the adenosine and nicotinamide portions, respect-
ively. The second step in Eqn 6 is energetically costly and
weakens the entire interaction to a remarkable extent.
Measurements of complex formation in vitro ledtothe
amazing conclusion that under saturating conditions less
than 20% of the nicotinamide is placed in contact with the
flavin, and is therefore available for hydride transfer [50].
The model of Fig. 3 explains why crystals of FNR with
bound analogues could be obtained with the wild-type
enzyme, whereas those involving NADP(H) complexes
were only possible with engineered FNR proteins in which
the carboxyl terminal tyrosine had been replaced by
nonaromatic residues such as serine [27]. In these FNR
mutants, rearrangement of the phenol group is no longer
required, and both pockets of the binding site would be
readily accessible.
Figure 4 shows amino acid residues involved in recogni-
tion of the adenosine-ribose and the NMN portions of the
dinucleotide, as identified through structural and mutage-
nesis studies [23,27,31,51]. They include residues displaying
charge interactions with the specific 2¢-phosphate group
presumably responsible for discrimination against NAD(H).
The increase in coenzyme affinity caused by substitutions of
the carboxyl terminal tyrosine was so dramatic that the
resulting FNR mutants were able to avidly incorporate
NADP
+
during biosynthesis in E. coli and keep it bound
through the purification and crystallization steps [27].
Incidentally, once the carboxyl terminal residue is replaced,

the electrostatic interactions at the 2¢-phosphate group of
NADP
+
are no longer sufficient to discriminate between
the coenzymes, and FNR becomes an efficient NAD(H)
oxidoreductase [50].
The reversible NADPH release depicted in step 9 also
represents the initial event of the reverse reaction, NADPH-
ferredoxin reductase, and will be addressed in further detail
in a forthcoming chapter, when discussing that FNR
activity.
Fig. 2. The electron transfer mechanism of ferredoxin-NADP(H)
reductase. The various steps of the catalytic pathway were initially
proposed by Batie and Kamin [60] on the basis of kinetic and binding
experiments on the spinach FNR. Oxidized forms are white, one-
electron reduced forms are light grey and two-electron reduced forms
are dark grey.
1904 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
Electron transfer from reduced ferredoxin
to ferredoxin-NADP(H) reductase
(Fig. 2, steps 2–4)
A recent article by Hurley et al. [57] has provided a very
comprehensive and updated review of experiments descri-
bing the interaction and electron transfer between Anabaena
FNR and Fd. Accordingly, the present section gives only a
concise account of these data; the reader is referred to the
above mentioned article for a more detailed description of
the two processes.
Conversion of oxidized FNR to the semiquinone form
(FNR

sq
) by reduced Fd (or Fld) is too fast to be measured
by rapid mixing techniques [44,60,65]. However, the kinetics
Fig. 3. Schematic representation of the bipartite NADP(H)-binding mode to ferredoxin–NADP(H) reductase. The model is based on the properties of
a tyrosine-to-serine site-directed mutant of pea FNR bound to NADP(H) [27,50]. Sites A and N represent the adenosine- and the nicotinamide-
binding regions, respectively, in the active site of FNR.
Table 2. Kinetic and binding parameters for various activities and interactions of ferredoxin-NADP(H) reductase. Binding and kinetic parameters
were averaged from experiments carried out at I £ 100 m
M
. (Original sources cited in the text.) When dispersion among reported values exceeded
50%, the interval between extreme data is indicated. ND, not determined.
Reaction
FNR source
K
m
or K
d
(l
M
) k
obs
or k
cat
(s
)1
)
Spinach leaves Anabaena E. coli Spinach leaves Anabaena E. coli
Binding
FNR
ox

– NADP
+
15 6 ND – – –
FNR
ox
–Fd
ox
(Fld
ox
) <1 4 (3)
a
0.5 (2)
a
–– –
Electron transfer
Fd
red
fi FNR fi NADP
+
10
b
ND ND 600 >200 ND
1
b
Fd
red
(Fld
red
) fi FNR
ox

ND 10 ND >600
c
60
c
6200 (>600)
c,a
250 (>600)
c,a
8 (25)
a
ND (8)
a
FNR
red
fi NADP
+
ND ND 500 >600 ND
NADPH fi FNR fi Fd
ox
fi cyt c 10
b
6
b
4
b
250 225 20
1
b
15
b

2
b
NADPH fi FNR fi Fld
ox
fi cyt c ND ND
33
b
4
b
7
b
ND 23–80 5
NADPH fi FNR
ox
<2 ND <5 >600
d
200
d
>600
d
>140
d
22
FNR
red
fi Fd
ox
(Fld
ox
) ND ND <5 (<5)

a
ND >600 (3)
a
2 (0.01)
a
NADPH fi FNR fi K
3
Fe(CN)
6
30
b
100
b
23
b
170
b
10
b
24
b
550 225–520 27
a
Values in parentheses are those obtained with Fld.
b
The upper and lower numerals indicate K
m
estimates for electron donors (Fd
red
,

NADPH) and acceptors (NADP
+
,Fd
ox
, Fld
ox
,K
3
Fe(CN)
6
), respectively.
c
The upper and lower values reported provide rates of transfer
for the first and second electron, respectively.
d
The upper and lower numerals show rates of formation of charge–transfer complexes and
rates of hydride transfer, respectively.
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1905
of this reaction could be resolved for the Anabaena
reductase by using laser flash photolysis, yielding a k
obs
of
about 6000 s
)1
and a K
d
of 1.7 l
M
for the transient FNR
ox

–
Fd
red
complex at I ¼ 100 m
M
[40,44,57]. Taking into
account the many contacts that are required to establish
this protein–protein interaction, it is somehow surprising
that FNR can efficiently accommodate Fd or Fld, two
proteins that differ completely in their primary, secondary
and tertiary structures. Other similarities must exist to
account for their functional equivalence, in spite of the lack
of homology. It is interesting to note, in this context, that
the molecular association of FNR with its electron partners
is steered by electrostatic interactions [37,40,47,56,66,67].
Using the Hodgkin index as a similarity measure, Ullmann
et al. [68] showed that Fd and Fld could be completely
overlapped on the basis of their surface electrostatic
potentials. The active sites and prosthetic groups of both
proteins, rather than their centres of mass, coincided in the
alignment [68].
Transient associations of the electron carriers in different
oxidation states are generally not amenable to structural
studies, but binary complexes of oxidized FNR and Fd
could be resolved by X-ray crystallography for both the
Anabaena and maize couples [29,69]. The resulting struc-
tures provided insightful data to complement chemical
cross-linking and mutagenesis studies, and helped to model
flavodoxin docking [46]. Fd binds to a concave region of the
FAD domain of maize FNR (Fig. 5A), burying an

accessible area of % 800 A
˚
2
in each partner, which repre-
sents about 5% and 15% of the total surface areas of FNR
and Fd, respectively [29]. The FAD and [2Fe)2S] redox
centres are sufficiently close (6.0 A
˚
) for direct electron
transfer through the space between the two prosthetic
groups (Fig. 5A–C). Distribution of surface charge and
calculations of the molecular dipole moments confirm the
relevance of complementary patches of basic and acidic
residues in FNR and Fd, respectively [66,67]. These polar
groups play a major role in determining the relative
orientation of the two electron carriers in the initial
nonproductive complex [40,66]. Attainment of the func-
tional conformations competent for electron exchange
requires further, fine adjustments, stabilized by a combina-
tion of well-defined hydrogen bonds, salt bridges, van der
Waals interactions and hydrophobic packing forces origin-
ating from the dehydration of the protein–protein interface
[40,47,48,52,57]. When the FNR molecules of the corn and
cyanobacterial complexes are superimposed, the Fd part-
ners appear rotated by an angle of 96°, indicating that many
protein–protein interactions are different in the two systems
[29,69]. Hurley et al. [57] have therefore proposed that, as in
the case of Fld binding, the crucial parameters selected
during evolution might be proximity of the prosthetic
groups in a nonpolar environment to facilitate direct

electron transfer.
Fig. 4. The nucleotide-binding site of ferredoxin–NADP(H) reductase. View of NADP(H) bound to the pea FNR-Y308S mutant reveals the intimate
interactions made by both the 2¢-P-AMP portion of the ligand and the nicotinamide. NADP(H) is depicted in blue, FAD in yellow, amino acids in
grey. Dashed lines mark interactions of £ 3.5 A
˚
thatmayengageinhydrogenbonds.ResiduesarelabelledasinpeaFNR(numbersinparentheses
are those of spinach FNR). The figure shows two water molecules that display hydrogen interactions with both NADP(H) and the protein
backbone. Modified from Deng et al. [27], drawn using
SWISS
-
PDBVIEWER
3.7 and rendered with
POV
-
RAY
.
1906 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
Full reduction of ferredoxin-NADP(H)
reductase (Fig. 2, steps 5–7)
When spinach FNR
ox
is mixed with excess Fd
red
in a
stopped flow system, all the flavoprotein is converted into
the semiquinone form in the dead time of the instrument
[60]. Transfer of the second electron, however, is too slow to
be compatible with steady-state catalysis, as already indica-
ted. The latter process actually involves various steps, which
are the dissociation of Fd

ox
(Fig. 2, step 4), binding of Fd
red
(step 5) and flavin reduction (step 6). The reaction is
strongly inhibited by Fd
ox
and stimulated by NADP
+
,
Fig. 5. Structure of the ferredoxin–NADP(H) reductase–ferredoxin complex. (A) View of the maize leaf bipartite FNR–Fd complex with the ribbon
diagram of Fd coloured in red, the FAD-binding domain of FNR in blue and the NADP(H) binding domain in pink. (B) Hypothetical tripartite
NADP(H)–FNR–Fd complex. View of the superposition of the maize leaf bipartite FNR–Fd complex and the pea FNR–NADP(H) complex. Pea
and maize FNR polypeptides were superimposed by the least square fitting of the isoalloxazine ring of FAD. The two domains of maize FNR are
shown in light blue, Fd in red, FAD in yellow and the NADP(H) from the pea FNR–NADP(H) crystals in deep blue. White arrows indicate the
[2Fe)2S] cluster (green), the isoalloxazine ring of FAD and the nicotinamide ring of NADP(H). (C) Superimposed view of the active site structure
of the maize FNR complexed with Fd (in red), the free maize FNR (in light blue) and the pea FNR complexed with NADP(H) (in grey). The
pyridine nucleotide is represented in blue. Note that in the three superimposed structures E312 (306) undergoes significant movements upon
complex formation. To facilitate the observation, E306 from pea FNR was omitted in the main figure and included in the inset. The models were
based on the detailed structures of the bipartite complexes reported by Kurisu et al. [29] and Deng et al. [27]. The figure was drawn using
SWISS
-
PDBVIEWER
3.7 and rendered with
POV
-
RAY
.
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1907
indicating that step 4 is the rate-limiting step, and that
NADP

+
facilitates Fd
ox
release, allowing the entire reaction
to proceed at a rapid pace through steps 4 and 8 [60]. The
sequence of events depicted in Fig. 2 agrees well with other
experimental observations. A ternary complex between the
two oxidized proteins and NADP
+
is readily formed
in vitro as measured by differential spectroscopy. The
affinity of FNR for Fd
ox
decreased > 10-fold on addition
of NADP
+
,andvice versa, indicating a strong case of
negative cooperativity for binding [59]. Essentially the same
results were obtained when using NADPH [59]. In this case,
dissociation of the two enzyme–product complexes, rather
than formation of the FNR–substrate species, are the most
demanding steps that limit turnover.
NADP
+
reduction and product release
(Fig. 2, steps 8 and 9)
Full FNR reduction renders a two-electron reduced Fd
ox
–
FNR

red
–NADP
+
complex that requires hydride transfer to
the nucleotide and dissociation to complete the catalytic
cycle. The sequence of steps proposed in Fig. 2 complies
with a canonical compulsory order mechanism, although
alternative pathways could be envisaged. For instance, it is
conceivable that Fd
ox
could dissociate from the FNR
red
–
NADP
+
complex prior to nucleotide reduction. Rapid
mixing experiments provided the main empirical support for
the proposed reaction order, showing that in a mixture of
the three components, the oxidation of FNR
red
byNADP
+
was faster than electron transfer from Fd
red
to the reductase
[60]. Even in the absence of Fd, NADP
+
reduction by
FNR
red

proceeds at % 500 s
)1
, a rate compatible with
catalysis [42,60].
Open question: the molecular bases of
cooperativity
As indicated previously, interactions of ferredoxin and
NADP
+
with FNR exhibit reciprocal negative cooperati-
vity, which is translated, paradoxically, into positive cooper-
ativity at the kinetic level [60,61]. It was expected therefore
that complex formation should lead to modifications in the
structure of the active site of the flavoprotein. Conforma-
tional movements resulting from nucleotide binding, how-
ever, appear to be largely restricted to displacement of the
carboxyl terminal tyrosine, as judged by the crystal structures
of wild-type and mutant FNR in complex with NADP(H)
and analogues [23,27,31]. We speculate that motion of the
phenol ring of tyrosine is responsible for the decrease in FNR
affinity for Fd, but the actual position of this residue when
pushed away by the entering nicotinamide is unknown. In a
tyrosine-to-tryptophan mutant of pea FNR that allows for
% 40% of nicotinamide occupancy, the displaced indole ring
failed to adopt a single ordered position [27].
Interaction of FNR and Fd, on the contrary, does lead
to structural changes in the two electron carriers relative to
the conformations of the free proteins [29]. On complex
formation, the NADP(H) domain is displaced slightly as a
single unit, and the side chain of Glu312 (numbering of

spinach FNR) moves to hydrogen bonding distance of the
hydroxyl group of Ser96 [29]. These two residues are highly
conserved among reductases of different origins, and their
charge, size and polarity are crucial to optimize the active
site geometry for electron and hydride transfer [38,43,
45,49]. The protein–protein interaction also affects the
microenvironments of the two prosthetic groups. The redox
potentials (E
m
) of Fd and FNR were shifted by )25 mV
and +20 mV, respectively, facilitating electron transfer in
the photosynthetic direction, namely, from Fd
red
to FNR
ox
[70].
It is not clear how these observed or putative structural
changes in the active site region of FNR correlate with the
induced-fit mechanism deduced from kinetic measurements.
Dorowski et al. [28] have proposed that Fd binding might
favour displacement of the carboxyl terminal tyrosine by
nestling the phenol group into a hydrophobic pocket of the
iron–sulphur protein. These authors advanced further and
challenged the model of Fig. 2 by suggesting that Fd is the
leader substrate that assists in NADP
+
binding. Although
such a mechanism would be at odds with the reported
decrease in NADP
+

affinity upon Fd attachment, the two
models can be reconciled by assuming that the dinucleotide
binds first in a nonproductive manner through its 2¢-P-AMP
portion. Fd may then interact with the tyrosyl residue,
favouring nicotinamide docking and establishing a loosely
bound complex compatible with turnover [28]. It is clear
that the carboxyl terminal tyrosine plays a pivotal role
during FNR catalysis, but further research will be required
to understand its actual function and importance.
The backward reaction is a mechanistic puzzle
Ferredoxin (or flavodoxin) reduction is the most widely
distributed function of FNR-type proteins. Table 1 pro-
vides a summary of metabolic routes that require such an
activity from either FNR or adrenodoxin reductase. In
cyanobacteria, a single FNR species functions as NADP
+
reductase in vegetative cells and as Fd reductase in
heterocysts [8], whereas two distinct isoforms fulfil these
roles in chloroplasts and nonphotosynthetic plastids of
vascular plants. In the latter case, tissue specificity is
determined at the transcriptional level by cis-acting regula-
tory elements [71,72]. Interestingly, the redox potentials of
these reductases and those of their corresponding ferredox-
ins have been tuned by evolution to favour the physiological
direction of electron transport [30]. However, the four
proteins can be readily exchanged in vitro when assayed in a
variety of reactions, indicating that the major force driving
NADP
+
or Fd reduction in vivo would be the availability of

substrates [30,73].
NADPH binding to oxidized FNR leads to rapid hydride
exchange between the nucleotide and the reductase, result-
ing in a succession of charge-transfer complexes involving
flavin and nicotinamide (Eqn 7, species in brackets), whose
formation can be followed by the appearance of long
wavelength absorbance signals [61].
FNR
ox
þ NADPH
!

FNR
ox
NADPH½
!

FNR
red
NADP
þ
ÂÃ
ð7Þ
The presence of various molecular species complicates the
quantitative estimation of binding equilibria. Batie and
Kamin [61] obtained an upper limit of about 2 l
M
for the
K
d

of the spinach FNR
ox
ÆNADPH complex, indicating that
1908 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
nucleotide binding to FNR
ox
is tighter in the reduced state.
Complex formation is very rapid (k
obs
> 500 s
)1
), followed
by slower hydride transfer to the flavin at 200 s
)1
(Table 2).
Electron transfer from FNR
red
to Fd
ox
istoofasttobe
followed by stopped-flow techniques [43,44,52]. All the
previous reactions proceed at velocities that are compatible
with the steady-state rate of Fd reduction (k
obs
¼ 200–
250 s
)1
), as measured by the cyt c reductase assay
[30,34,35,43,44,52,57].
The reversible nature of the various steps involved in

FNR-mediated NADP
+
reduction suggested that electron
transfer from NADPH to Fd should proceed by a reversed
version of the ordered pathway of Fig. 2. The forward and
backward reactions are shown, in Cleland’s notation, in
Fig. 6A,B, respectively. Assuming that v ¼ k
)6
[FNR
ox
]
[NADPH] ) k
6
[FNR
ox
ÆNADPH + FNR
red
ÆNADP
+
], then
in the absence of products the velocity equation for Fd
reduction (Fig. 6B) will be:
Where K
m
, K
d
and V
m
have their conventional meanings
and K

m
¢
(Fd)
represents the sum of the K
m
values for the
successive interactions of the two molecules of Fd
ox
with
FNR
red
and FNR
sq
(Fig. 6B). Equation 8 predicts that
double reciprocal plots of v against the concentrations of any
of the two substrates should yield straight lines intersecting
in the fourth quadrant, as it occurs with the forward
reaction. There is no simple way to measure Fd (or Fld)
reduction, because the reduced acceptor is reoxidized by
dissolved oxygen with k
cat
% 40 s
)1
[16]. Therefore, kinetic
evaluation of the Fd reductase activity requires measure-
ments under strict anaerobiosis, and experiments of this kind
have not yet been carried out with the plant-type flavopro-
teins. As indicated before, cytochrome c is usually employed
as a final acceptor that competes favourably with dioxygen
for the spare electron of reduced Fd (Eqns 4 and 5). In vivo,

the physiological acceptor enzymes (such as thioredoxin
reductase, nitrate reductase or dihydroascorbate reductase)
would play a similar role, preventing accumulation of the
reduced ÔdoxinsÕ that, under the oxygen tensions prevailing
in aerobic cells, could otherwise facilitate the propagation of
superoxide radicals and other toxic oxygen derivatives.
Wan and Jarrett [63] have measured the anaerobic
oxidation of NADPH by an E. coli system made up of
FNR and either ferredoxin or any of the two bacterial
flavodoxins. This reductase species is remarkably slow,
turning over at 0.15 s
)1
for Fd and about 0.004 s
)1
for the
flavodoxins [63]. The rates of individual electron transfer
steps are consistent with this slow pace of catalysis ([62,63],
summarized in Table 2). Moreover, E. coli FNR mediates
direct reduction of cytochrome c at % 5s
)1
,withthisrate
being enhanced only about twofold by the addition of
saturating flavodoxin [62]. Similar low activities have been
obtained with the A. vinelandii [74] and Rhodobacter
capsulatus (C. Bittel, N. Carrillo and N. Cortez, IBR,
Rosario, Argentina, unpublished observations) reductases.
The collected results indicate that these bacterial FNR forms
display distinct catalytic features, and their comparison with
the plant-type enzymes needs to be considered with caution.
Surprisingly, when spinach Fd reduction was measured

in vitro by the cyt c assay, parallel lines were obtained in 1/v
vs. 1/[NADPH] plots, suggesting a two-step transfer Ôping-
pongÕ mechanism without formation of a ternary complex
[75]. The diaphorase activity (with various electron partners)
also conforms to a double-displacement mechanism [75,76],
although in this case the electronic route between the flavin
Fig. 6. The forward and reverse reactions
catalysed by ferredoxin-NADP(H) reductase.
NADP
+
reduction (A) follows the compul-
sory ordered pathway of Fig. 2, whereas two
alternative mechanisms are proposed for
electron transfer in the reverse direction:
ordered (B), or two-step transfer (C). E,
FNR
ox
;F,FNR
sq
;G,FNR
red
;A,NADP
+
P, NADPH; B, Fd
red
;Q,Fd
ox
.
m ¼
NADPH½Fd

ox
½V
m
K
dðNADPHÞ
K
mðNADPHÞ
þ K
0
mðFdÞ
NADPH½þK
mðNADPHÞ
Fd
ox
½þ½NADPH½Fd
ox

ð8Þ
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1909
and the artificial acceptor might well be different from that
involving the natural substrate. Then, either the presence of
the coupled cytochrome in the Fd reduction assay modifies
the reaction pathway, or the ordered mechanism of FNR
has some kind of anomalous kinetics. In fact, there are a
number of situations that could produce such behaviour
[77]. For example, Massey (quoted in [75]) has shown that a
catalytic pathway similar to that of Fig. 2 can still produce
ping-pong kinetics depending on the rates of ternary
complex formation and dissociation relative to those of
electron and/or hydride transfer. In the case of FNR, this

would imply that FNR
ox
ÆNADPH + Fd
ox
are rapidly
converted into FNR
ox
ÆNADP
+
+Fd
red
(Fig. 2, steps 8–
2). Unfortunately, these intermediate steps are not accessible
to steady-state or rapid reaction measurements. An alter-
native explanation for the anomalous kinetic behaviour of
the ferredoxin reductase activity could be that the FNR
red
Æ
NADP
+
complex is dissociated prior to Fd
ox
binding,
yielding an entirely ping-pong mechanism (Fig. 6C). If, as
before, v ¼ k
-6
[FNR
ox
][NADPH] ) k
6

[FNR
ox
ÆNADPH
+FNR
red
ÆNADP
+
], then Eqn 9 predicts parallel straight
lines for all substrates in double reciprocal plots.
The definitions of K
m
, V
m
and K
m
¢
(Fd)
are those of Eqn 8.
This model has several features that can be readily put to
test, including the dependence of v on [Fd
ox
]. Even though
such an experiment has not been reported, replotting the
original data of Forti and Sturani [75] shows that their
results indeed conform to a double-displacement mecha-
nism as depicted in Fig. 6C. Product inhibition studies
could also be especially revealing to diagnose the kinetic
mechanism, as the product of the reaction, NADP
+
,is

expected to display mixed-type inhibition with respect to
NADPH, instead of the competitive inhibition predicted by
the compulsory ordered pathway of Fig. 6B.
Preference for different pathways in the forward and
reverse reactions of FNR suggests that distinct kinetic
constraints are present on one or both of these routes. As
indicated before, formation of ternary complexes during
NADP
+
reduction is required to ensure Fd
ox
release after
electron transfer (Fig. 2, steps 4 and 8). We propose that
this requirement would be relieved in the reverse reaction
if Fd
red
dissociates from FNR
sq
and FNR
ox
at rates
compatible with steady-state catalysis. This proposal gains
support from a number of fast kinetic determinations
[43,44,52], although a comprehensive description of the
relevant reaction steps would require determination of
very high rates of electron transfer in binary and ternary
complexes.
Open question: the inhibitory effect of
NADPH
FNR-dependent activities are inhibited by a brief incuba-

tion of the reductase with NADPH [76]. Several lines of
evidence indicate that inactivation depends on electron
transfer from the reduced pyridine nucleotide to the FAD
group. The wild-type enzyme is insensitive to NADH, but
the tyrosine mutant that has become proficient as NADH
oxidoreductase is readily inactivated by this nucleotide
(E. Massa, V. Rapisarda, E. Ceccarelli and N. Carrillo,
IBR, Rosario, Universidad de Tucuma
´
n, Argentina,
unpublished results). The effect of NADPH can be preven-
ted and reversed by NADP
+
and by FNR acceptors,
including O
2
[64,76]. Conversely, anaerobic incubation with
the pyridine nucleotide accelerated the loss of enzymatic
activity, suggesting that flavin reduction to the hydroqui-
none level was a mandatory feature of inactivation [64].
In the presence of 4
M
urea, a concentration that by itself
has little effect on spinach FNR function, the reversible
NADPH inhibition is turned into an irreversible one, where
FAD becomes accessible to phosphodiesterase and eventu-
ally splits from the apoprotein [76]. Also, heating of E. coli
FNR with excess NADPH under anaerobic conditions
results in rapid denaturation of the enzyme, with a T
m

of
41 °CandDH ¼ 80 kcalÆmol
)1
, compared to 66 °Cand
100 kcalÆmol
)1
for the oxidized reductase [64]. Chemical
reduction of the flavoprotein with dithionite produced
essentially the same melting curve, indicating that FNR
inactivation was due to a poor interaction between the
reduced cofactor and the apoprotein, irrespective of the
source of reducing equivalents [64].
The results suggest that one or more of the reactions
started by NADPH binding to FNR
ox
(Eqn 7) leads to a
major destabilization of the holoenzyme. None of this could
be appreciated, however, when analysing the atomic struc-
ture of a complex between NADPH and the tyrosine-
to-serine mutant of pea FNR. Most interactions established
by the reduced dinucleotide were identical to those displayed
byNADP
+
[27]. Crystals soaked aerobically with excess
NADPH shifted from golden yellowto blue-green, indicating
the formation of charge-transfer complexes and probably
also some FNR
sq
, as electrons were slowly conveyed, one at a
time, to molecular oxygen [27]. Conformational changes

could also be a direct consequence of FAD reduction to the
hydroquinone state. However, the structure of a dithionite-
reduced reductase failed to show significant differences with
respect to that of the oxidized flavoprotein [23]. The
molecular mechanisms underlying the inactivating and
destabilizing effects of NADPH remain therefore as obscure
as they were 35 years ago, when described for the first time.
Open question: the best possible ferredoxin-
NADP(H) reductase
This chapter deals with the efficiency of FNR as a catalyst
and with the requirements for catalytic improvement. The
topic is not entirely devoid of practical meaning, because
FNR has been shown to mediate the rate-limiting step in
photosynthesis under both saturating and limiting light
regimes [78]. In addition, the reductases from E. coli,
A. vinelandii, and presumably other bacteria, display turn-
over numbers that are 20- to 100-fold lower than those of
their plastidic and cyanobacterial counterparts, with whom
they share extensive structural and conformational identity
m ¼
NADPH½Fd
ox
½V
m
K
0
mðFdÞ
NADPH½þK
mðNADPHÞ
Fd

ox
½þNADPH½Fd
ox
½
ð9Þ
1910 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
[20–23,27–33]. At the other extreme of the spectrum,
Piubelli et al. [50] have described a site-directed mutant of
pea FNR whose carboxyl terminal tyrosine was replaced by
phenylalanine (Y308F FNR), resulting in a sevenfold
increase of catalytic competence for NADPH in the
ferricyanide-dependent diaphorase reaction. The k
cat
/K
m
ratio, usually employed as a quantitative estimation of the
catalytic efficiency [79], was shown to increase from
6 l
M
)1
Æs
)1
in E. coli FNR to 17 l
M
)1
Æs
)1
in the wild-type
pea enzyme, and to 155 l
M

)1
Æs
)1
in the Y308F mutant
[50,63]. Therefore, it might conceivably be asked why this
optimized reductase version with the phenylalanine replace-
ment has not been selected during evolution and instead,
that a tyrosine residue is strongly conserved at the corres-
ponding position in the vast majority of FNR proteins
described so far.
Catalytic competence is obviously not the only trait that
determines the evolutionary success of an enzyme. In their
natural media, selective forces might operate to optimize the
cost of synthesis and folding, the stability toward denatur-
ation and proteolysis, or even the route to its subcellular
destiny. However, the importance of efficiency for an
enzyme is equally evident. In principle, a catalyst can
increase this efficiency to approximate that of diffusion-
controlled processes, that is, reactions whose rate-limiting
steps are the diffusion of reagents to the active site. Several
enzymes, such as carbonic anhydrase or triosephosphate
isomerase, are close to that upper limit [79]. Assessment of
the evolution of enzymes to catalytic perfection also requires
efficiency to be related with the context in which the enzyme
operates, either in vivo or in vitro, and including the levels of
substrates, products and activity modulators. Albery and
Knowles [80] have introduced an efficiency function and a
formalism to describe the effectiveness of a catalyst in
accelerating a chemical reaction under various conditions. A
flux parameter Y is defined as the ratio of the enzyme

concentration over the velocity of the process it catalyses:
Y ¼½E=m ð10Þ
Using the steady-state approximation, the value of Y can be
related to the kinetic parameters of any catalytic process.
Assumptions have to be made on whether the reaction is
reversible or irreversible, involves one or more substrates,
inhibitors, activators, etc. As an example, the expression of
Y for a reversible reaction involving one substrate, and not
affected by inhibitors or activators, is given below:
Y ¼ K
m
=k
cat
Á½Sþ1=k
cat
þ h=k
0
cat
ð11Þ
Where K
m
and k
cat
represent the kinetic parameters of the
forward reaction, k¢
cat
the rate constant of the reverse
reaction, and the value of h is one or zero for reversible or
irreversible processes, respectively [80]. Equation 11 indi-
cates that Y is a function of the catalytic properties of the

enzyme and the actual substrate concentration [S], which is
the only environmental constraint affecting efficiency in the
example provided. The Y-value also has a minimum limit,
Y
m
, for the perfect catalyst.
Y
m
¼ 1=k
dif
Á½Sþ1=K
e
Ák
dif
½Sð12Þ
Where k
dif
is the rate constant for the diffusion-controlled
process and K
e
is the overall equilibrium constant for the
interconversion between substrate and product. The effi-
ciency function E
f
can now be defined as
E
f
¼ Y
m
=Y ð13Þ

The value of E
f
for a given substrate will approach unity as
the enzyme becomes a more efficient catalyst. Using the
kinetic parameters of Table 2 and k
dif
% 5 · 10
5
M
)1
Æs
)1
[79], we estimate that E
f
for electron transfer between
photosynthetic FNR and NADP(H) is about 10
)2
at a
nucleotide concentration of 0.1 m
M
. Under identical con-
ditions, the E
f
of the E. coli flavoprotein is only 5 · 10
)4
.
Because most enzymes characterized so far have E
f
values in
the range of 10

)4
)10
)3
[79], chloroplast FNR must be
regarded as a quite proficient reductase in vivo.
We then asked how this catalytic competence could still
be enhanced, either by increasing k
cat
, decreasing K
m
or
both. To determine whether the phenylalanine substitution
of the pea reductase with optimized k
cat
/K
m
ratio represents
a real advantage under physiological conditions, we studied
the behaviour of E
f
as a function of [NADPH] for this
mutant enzyme and its wild-type parental. We chose the
ferricyanide reduction assay because this is the only FNR
activity described to date in which hydride transfer is the
rate-limiting step (Table 2). In addition, the reaction is
basically irreversible, so that the last terms in the right-hand
sides of Eqns (11) and (12) cancel out, and the E
f
function
reduces to:

E
f
¼ k
cat
=k
dif
ðK
m
þ½NADPHÞ ð14Þ
The results of plotting E
f
as a function of NADPH
concentrations are shown in Fig. 7A. The Y308F mutant
is indeed more proficient at low [NADPH], but as substrate
amounts are raised this advantage fades away rapidly and at
the nucleotide levels found in most cells and plastids (100–
500 l
M
), the two enzymes have similar efficiencies (Fig. 7A,B).
This example illustrates the general principle that, contrary
to common intuition, enzymes evolve by maximizing k
cat
/
K
m
while keeping K
m
‡ [S]. With respect to the affinity for
NADP(H), the plant FNR has already reached its maximal
potential in relation to its cellular environments, and any

further improvement will need to be through increases in
k
cat
. The tyrosine-to-phenylalanine mutant, which traded
off substrate affinity for thermodynamic stability [39,50],
does not represent a better enzyme in vivo, compared to the
wild-type.
A similar comparison made with the reductases from pea
and E. coli provides an interesting counterexample.
Although the plant flavoenzyme is always more efficient
than its bacterial homologue, the differences are relatively
small (approximately threefold) at low substrate concentra-
tion, increasing sharply with the nucleotide level to attain an
upper limit of about 20-fold above 100 l
M
NADPH
(Fig. 7B). It has been argued that the low rate of hydride
transfer displayed by E. coli FNR is caused by the stacking
interactions between Tyr247 and Trp248 with the isoallox-
azine and adenine ring systems, that are expected to shield
the NADP(H) binding site and impede easy access of the
cofactor [62–64]. However, the affinity for the pyridine
nucleotide is actually higher in the bacterial FNR (Table 2).
Furthermore, the analysis of Fig. 7 indicates that develop-
ment of catalytic competence by this enzyme would require
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1911
a significant increase in k
cat
. Replacement of the carboxyl
terminal aromatic residues of E. coli FNR would probably

result in higher affinity for the nucleotide, as it occurs in the
pea enzyme [50], a modification that is not expected to
confer selective advantage. In general, ferredoxin reductases
have K
m
values for NADP(H) that are well below the
physiological concentrations of this coenzyme and there-
fore, any further decrease is irrelevant in catalytic terms. It is
evident that the FNR proteins of the E. coli group have
constraints on electron transfer that are relieved in their
Anabaena and plant counterparts, but it is unlikely that
these constraints are related to faulty access to the
nucleotide binding site.
Finally, the Haldane equations establish that the value of
k
cat
/K
m
cannot reach the diffusion-controlled limit for a
reaction that is thermodynamically unfavourable [79].
Therefore, the E
m
values of plant FNR isoforms predict
that the activity of the photosynthetic reductases could only
be optimized in the direction of NADP
+
reduction and
those of the heterotrophic flavoproteins, in the direction of
Fd reduction.
Conclusions and prospects

Ferredoxin-NADP(H) reductases display a number of
outstanding properties. They are stable, ubiquitous and
can be readily overproduced in microorganisms. Their
two-domain structure constitutes the basic framework for
many other electron transfer flavoproteins that share with
FNR, besides the overall conformational design, similar
modes of ligand binding and common pathways of
electron and hydride transfer. Over the last few years,
investigations on the structure, physiological function and
natural history of FNR have experienced remarkable
progress, but despite these considerable advances, several
key features of FNR enzymology remain unsolved. The
recognition of the molecular bases of induced fit will
require refined structural studies under dynamic condi-
tions, particularly concerning the seemingly all-important
role of the carboxyl terminal tyrosine. High-resolution
spectroscopic methods such as nuclear magnetic resonance
or time-resolved fluorescence measurements, applied on
both wild-type and site-directed mutants of the reductase,
appear as especially promising in providing new clues to
understand this key feature of FNR catalysis. Explanation
of the reductive inactivation of the flavoenzyme, on the
other hand, awaits a more detailed description of the
chemistry of the process, combined with functional and
conformational studies. Finally, it is necessary to formulate
comprehensive kinetic models for both the forward and
reverse reactions. In the latter case, and even though there
is still much to be done using the cyt c assay, especially
with respect to product inhibition analysis, studies on the
direct reduction of Fd under anaerobic conditions need to

be pursued with more vigour.
In addition to their involvement in photosynthesis and
other pathways of central metabolism, flavoenzymes with
FNR activity can participate in a number of secondary
processes that might hold biotechnological and/or pharma-
ceutical relevance, such as reductive detoxification of
xenobiotics, activation of oncogenic drugs and elimination
of oxygen-centred radicals [14,18,19]. Modification of the
catalytic efficiency of FNR is then significant on both
scientific and technological grounds, and our calculations
indicate that there are ample possibilities for optimization
of its activity. The availability of FNR-encoding DNA
sequences from many different sources makes this reductase
accessible to directed evolution through gene shuffling or
error-prone recombination. In the present article, we
elaborated some general principles pertinent to FNR
enzymology that might facilitate this task, based on the
actual conditions at which the enzyme would have to
operate in vivo or in vitro.
Fig. 7. Variation of the efficiency function E
f
with the concentration of
NADPH for wild-type and mutant ferredoxin-NADP(H) reductases.
(A) Calculated enzymatic efficiencies (E
f
) as a function of the NADPH
concentration. Dashed line, pea FNR-Y308F; thin solid line, wild-type
pea FNR; thick solid line, E. coli FNR. (B) The E
f
ratios between

FNR–Y308F and wild-type pea FNR (dashed line), and between wild-
type pea and E. coli FNR (solid line), are plotted as a function of
NADPH concentration. The construction and general properties of a
site-directed mutant of pea FNR carrying a phenylalanine substitution
at the carboxyl terminal position have been described elsewhere
[36,50]. Calculations were made for the ferricyanide-dependent
diaphorase activity as indicated in the text. The shaded area represents
the range of NADPH concentrations normally found in plastids and
cells (for instance, a value of 150 l
M
NADPH for the E. coli cytosol
has been reported by Penfound and Foster [81]).
1912 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
Acknowledgements
NC and EAC are staff members of the Consejo Nacional de
Investigaciones Cientı
´
ficas y Te
´
cnicas (CONICET, Argentina). This
study was supported by grants from Fundacio
´
n Antorchas, CONICET
and Agencia de Promocio
´
nCientı
´
fica y Tecnolo
´
gica (ANPCyT,

Argentina).
References
1. Avron, M. & Jagendorf, A.T. (1956) A TPNH diaphorase from
chloroplasts. Arch. Biochem. Biophys. 65, 475–490.
2. Shin, M. & Arnon, D.I. (1965) Enzymic mechanisms of pyridine
nucleotide reduction in chloroplasts. J. Biol. Chem. 240, 1405–
1411.
3. Dudley,K.H.,Ehrenberg,A.,Hemmerich,P.&Mu
¨
ller, F. (1964)
Spektren und Strukturen der am Flavin-Redoxsystem beteiligten
Partikeln. Helv. Chim. Acta 47, 1354–1383.
4. Jungermann,K.,Kirchniawy,H.,Katz,N.&Thauer,R.K.(1974)
NADH, a physiological electron donor in clostridial nitrogen
fixation. FEMS Microbiol. Lett. 43, 203–206.
5. Chen, Y.P. & Yoch, D.C. (1989) Isolation, characterization and
biological activity of ferredoxin-NAD
+
reductase from the
methane oxidizer Methylosinus trichosporium OB3b. J. Bacteriol.
171, 5012–5016.
6. Hanukoglu, I. & Gutfinger, T. (1989) cDNA sequence of adreno-
doxin reductase. Identification of NADP
+
-binding sites in
oxidoreductases. Eur. J. Biochem. 180, 479–484.
7. Bianchi, V., Hagga
˚
rd-Ljungquist,E.,Pontis,E.&Reichard,P.
(1995) Interruption of the ferredoxin (flavodoxin) NADP

+
oxidoreductase gene of Escherichia coli does not affect anaerobic
growth but increases sensitivity to paraquat. J. Bacteriol. 177,
4528–4531.
8. Razquin, P., Fillat, M.F., Schmitz, S., Stricker, O., Bo
¨
hme, H.,
Go
´
mez-Moreno, C. & Peleato, M.L. (1996) Expression of ferre-
doxin-NADP
+
reductase in heterocysts from Anabaena sp. Bio-
chem. J. 316, 157–160.
9. Lacour, T. & Dumas, B. (1996) A gene encoding a yeast equivalent
to mammalian NADPH-adrenodoxin oxidoreductases. Gene 174,
289–292.
10. Vollmer, M., Thomsen, N., Wiek, S. & Seeber, F. (2001) Api-
complexan parasites possess distinct nuclear-encoded, but apico-
plast-localized, plant-type ferredoxin-NADP
+
reductase and
ferredoxin. J. Biol. Chem. 276, 5483–5490.
11. Carrillo, N. (1998) Partition of ferredoxin-NADP
+
oxidoreduc-
tase between photosynthesis and antioxidant metabolism. In
Photosynthesis: Mechanisms and Effects (Garab, G., ed.), Vol. 3,
pp. 1511–1516. Kluwer, Dordrecht, the Netherlands.
12. Van Thor, J.J., Jeanjean, R., Havaux, M., Sjollema, K.A.,

Joset, F., Hellingwerf, K.J. & Matthijs, H.C.P. (2001) Salt shock-
inducible photosystem I cyclic electron transfer in Synechocystis
PCC6803 relies on binding of ferredoxin-NADP
+
reductase to
the thylakoid membrane via its CpcD phycobilisome-linker
homologous N-terminal domain. Biochim. Biophys. Acta 1457,
129–144.
13. Zheng,M.,Doan,B.,Schneider,T.D.&Storz,G.(1999)OxyR
and SoxRS regulation of fur. J. Bacteriol. 181, 4639–4643.
14. Krapp, A.R., Rodriguez, R.E., Poli, H.O., Paladini, D.H.,
Palatnik, J.F. & Carrillo, N. (2002) The flavoenzyme ferredoxin
(flavodoxin)-NADP(H) reductase modulates NADP(H) home-
ostasis during the soxRS response of Escherichia coli. J. Bacteriol.
184, 1474–1480.
15. Carrillo, N. & Vallejos, R.H. (1987) Ferredoxin-NADP
+
reductase. In The Light Reactions. Topics in Photosynthesis
(Barber, J., ed.), Vol. 8, pp. 527–560. Elsevier, Amsterdam,
the Netherlands.
16. Go
´
mez-Moreno, C. & Bes, M.T. (1994) Structural requirements
for the electron transfer between a flavoprotein and viologens.
Biochim. Biophys. Acta 1187, 236–240.
17. Massey, V. (1994) Activation of molecular oxygen by flavins and
flavoproteins. J. Biol. Chem. 269, 22459–22462.
18. Shah, M.M. & Spain, J.C. (1996) Elimination of nitrite from the
explosive 2,4,6-trinitrophenylmethylnitramine (tetryl) catalyzed by
ferredoxin NADP

+
oxidoreductase from spinach. Biochim. Bio-
phys. Res. Commun. 220, 563–568.
19. Kranendonk, M., Commandeur, J.N., Laires, A., Rueff, J. &
Vermeulen, N.P. (1997) Characterization of enzyme activities and
cofactors involved in bioactivation and bioinactivation of chemi-
cal carcinogens in the tester strains Escherichia coli K12 MX100
and Salmonella typhimurium LT2 MA100. Mutagenesis 12, 245–
254.
20. Karplus, P.A., Daniels, M.J. & Herriott, J.R. (1991) Atomic
Structure of ferredoxin-NADP
+
reductase: prototype for a
structurally novel flavoenzyme family. Science 251, 60–66.
21. Correll, C.C., Ludwig, M.L., Bruns, C.M. & Karplus, P.A. (1993)
Structural prototypes for an extended family of flavoprotein
reductases. Comparison of phthalate dioxygenase reductase with
ferredoxin reductase and ferredoxin. Protein Sci. 2, 2112–2133.
22. Karplus, P.A. & Bruns, C.M. (1994) Structure–function relations
for ferredoxin reductase. J. Bioenerg. Biomembr. 26, 89–99.
23. Bruns, C.M. & Karplus, P.A. (1995) Refined crystal structure of
spinach ferredoxin-NADP
+
oxidoreductase at 1.7 A
˚
resolution:
oxidized, reduced and 2¢-phospho-5¢-AMP bound states. J. Mol.
Biol. 247, 1125–1145.
24. Lu,G.,Campbell,W.H.,Schneider,G.&Ljundquist,Y.(1994)
Crystal structure of the FAD-containing fragment of corn nitrate

reductase at 2.5 A
˚
resolution. Relationship to other flavoprotein
reductases. Structure 2, 809–821.
25. Wang, M., Roberts, D.L., Paschke, R., Shea, T.M., Masters, R.S.
& Kim, J.J. (1997) Three-dimensional structure of NADPH-
cytochrome P450 reductase. Prototype for FMN- and FAD-
containing enzymes. Proc. Natl Acad. Sci. USA 94, 8411–8416.
26. Gruez, A., Pignol, D., Zeghouf, M., Cove
`
s, J., Fontecave, M.,
Ferrer, J.L. & Fontecilla-Camps, J.C. (2000) Four crystal struc-
tures of the 60 kDa flavoprotein monomer of the sulfite reductase
indicate a disordered flavodoxin-like module. J. Mol. Biol. 299,
199–212.
27. Deng, Z., Aliverti, A., Zanetti, G., Arakaki, A.K., Ottado, J.,
Orellano, E.G., Calcaterra, N.B., Ceccarelli, E.A., Carrillo, N. &
Karplus, P.A. (1999) A productive NADP
+
binding mode of
ferredoxin-NADP
+
reductase revealed by protein engineering and
crystallographic studies. Nat. Struct. Biol. 6, 847–853.
28. Dorowski, A., Hofmann, A., Steeghorn, C., Boicu, M. & Huber,
R. (2001) Crystal structure of paprika ferredoxin-NADP
+
reductase. Implications for the electron transfer pathway. J. Biol.
Chem. 276, 9253–9263.
29. Kurisu, G., Kusunoki, M., Katoh, E., Yamazaki, T., Teshima, K.,

Onda, Y., Kimata-Ariga, Y. & Hase, T. (2001) Structure of the
electron transfer complex between ferredoxin and ferredoxin-
NADP
+
reductase. Nat. Struct. Biol. 8, 117–121.
30. Aliverti, A., Faber, R., Finnerty, C.M., Ferioli, C., Pandini, V.,
Negri, A., Karplus, P.A. & Zanetti, G. (2001) Biochemical and
crystallographic characterization of ferredoxin-NADP
+
reductase
from nonphotosynthetic tissues. Biochemistry 40, 14501–14508.
31. Serre, L., Vellieux, F.M.D., Medina, M., Go
´
mez-Moreno, C.,
Fontecilla-Camps, J.C. & Frey, M. (1996) X-ray structure of the
ferredoxin-NADP
+
reductase from the cyanobacterium Anabaena
PCC7119 at 1.8 A
˚
resolution, and crystallographic studies of
NADP
+
binding at 2.25 A
˚
resolution. J. Mol. Biol. 263, 20–39.
32. Ingelmann, M., Bianchi, V. & Eklund, H. (1997) The three-
dimensional structure of flavodoxin reductase from Escherichia
coli at 1.7 A
˚

resolution. J. Mol. Biol. 268, 147–157.
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1913
33. Sridhar Prasad, G., Kresge, N., Muhlberg, A.B., Shaw, A., Jung,
Y.S.,Burgess,B.K.&Stout,C.D.(1998)Thecrystalstructureof
NADPH:ferredoxin reductase from Azotobacter vinelandii. Pro-
tein Sci. 7, 2541–2549.
34. Aliverti, A., Lu
¨
bberstedt, T., Zanetti, G., Herrmann, R.G. &
Curti, B. (1991) Probing the role of Lysine 116 and Lysine 224 in
the spinach ferredoxin-NADP
+
reductase by site-directed muta-
genesis. J. Biol. Chem. 266, 17760–17763.
35. Aliverti, A., Piubelli, L., Zanetti, G., Lu
¨
bberstedt, T., Herrmann,
R.G. & Curti, B. (1993) The role of cysteine residues of spinach
ferredoxin-NADP
+
reductase as assessed by site-directed muta-
genesis. Biochemistry 32, 6374–6380.
36. Orellano, E.G., Calcaterra, N.B., Carrillo, N. & Ceccarelli, E.A.
(1993) Probing the role of the carboxyl terminal region of plant
ferredoxin-NADP
+
reductase by site-directed mutagenesis and
deletion analysis. J. Biol. Chem. 268, 19267–19273.
37. Aliverti, A., Corrado, M.E. & Zanetti, G. (1994) Involvement of
lysine-88 of spinach ferredoxin-NADP

+
reductase in the inter-
action with ferredoxin. FEBS Lett. 343, 247–250.
38. Aliverti, A., Bruns, C.M., Pandini, V.E., Karplus, P.A., Vanoni,
M.A.,Curti,B.&Zanetti,G.(1995)Involvementofserine96in
the catalytic mechanism of ferredoxin-NADP
+
reductase: struc-
ture-function relationship as studied by site-directed mutagenesis
and X-ray crystallography. Biochemistry 34, 8371–8379.
39. Calcaterra, N.B., Pico
´
, G.A., Orellano, E.G., Ottado, J., Carrillo,
N. & Ceccarelli, E.A. (1995) Contribution of the FAD binding site
residue tyrosine 308 to the stability of pea ferredoxin-NADP
+
oxidoreductase. Biochemistry 34, 12842–12848.
40. Hurley, J.K., Fillat, M.F., Go
´
mez-Moreno, C. & Tollin, G. (1996)
Electrostatic and hydrophobic interactions during complex for-
mation and electron transfer in the ferredoxin/ferredoxin-NADP
+
reductase system from Anabaena. J. Am. Chem. Soc. 118, 5526–
5531.
41. Martı
´
nez-Ju´ lvez, M., Hurley, J.K., Tollin, G., Go
´
mez-Moreno, C.

& Fillat, M.F. (1996) Overexpression in E. coli of the complete
petH gene product from Anabaena: purification and properties of
a 49 kDa ferredoxin- NADP
+
reductase. Biochim. Biophys. Acta
1297, 200–206.
42. Arakaki, A.K., Ceccarelli, E.A. & Carrillo, N. (1997) Plant-type
ferredoxin-NADP
+
reductases: a basal structural framework and
a multiplicity of functions. FASEB J. 11, 133–140.
43. Medina, M., Martı
´
nez-Ju´ lvez, M., Hurley, J.K., Tollin, G. &
Go
´
mez-Moreno, C. (1998) Involvement of Glutamic 301 in the
catalytic mechanism of ferredoxin-NADP
+
reductase from
Anabaena PCC 7119. Biochemistry 37, 2715–2728.
44. Martı
´
nez-Ju´ lvez,M.,Hermoso,J.,Hurley,J.K.,Mayoral,T.,
Sanz-Aparicio, J., Tollin, G., Go
´
mez-Moreno, C. & Medina, M.
(1998) Role of Arg100 and Arg264 from Anabaena ferredoxin-
NADP
+

reductase for optimal NADP
+
binding and electron
transfer. Biochemistry 37, 17680–17691.
45. Aliverti, A., Deng, Z., Ravasi, D., Piubelli, L., Karplus, P.A. &
Zanetti, G. (1998) Probing the function of the invariant glutamyl
residue 312 in spinach ferredoxin-NADP
+
reductase. J. Biol.
Chem. 273, 34008–34015.
46. Martı
´
nez-Ju´ lvez,M.,Medina,M.&Go
´
mez-Moreno, C. (1999)
Ferredoxin-NADP
+
reductase uses the same site for the inter-
action with ferredoxin and flavodoxin. J. Biol. Inorg. Chem. 4,
568–578.
47. Hurley, J.K., Hazzard, J.T., Martı
´
nez-Ju´ lvez, M., Medina, M.,
Go
´
mez-Moreno, C. & Tollin, G. (1999) Electrostatic forces
involved in orienting Anabaena ferredoxin during binding to
Anabaena ferredoxin: NADP
+
reductase: site-specific mutagenesis,

transient kinetic measurements, and electrostatic surface. Protein
Sci. 8, 1614–1622.
48. Hurley, J.K., Faro, M., Brodie, T.B., Hazzard, J.T., Medina, M.,
Go
´
mez-Moreno, C. & Tollin, G. (2000) Highly nonproductive
complexes with Anabaena ferredoxin at low ionic strength are
induced by nonconservative amino acid substitution at Glu139 in
Anabaena ferredoxin-NADP
+
reductase. Biochemistry 39, 13695–
13702.
49. Mayoral, T., Medina, M., Sanz-Aparicio, J., Go
´
mez-Moreno, C.
& Hermoso, J. (2000) Structural basis of the catalytic role of
Glu301 in Anabaena PCC 7119 ferredoxin-NADP
+
reductase
revealed by X-ray crystallography. Proteins 38, 60–69.
50. Piubelli, L., Aliverti, A., Arakaki, A.K., Carrillo, N., Ceccarelli,
E.A.,Karplus,P.A.&Zanetti,G.(2000)Competitionbetween
C-terminal tyrosine and nicotinamide modulates pyridine
nucleotide affinity and specificity in plant ferredoxin-NADP
+
reductase. J. Biol. Chem. 275, 10472–10476.
51. Medina, M., Luquita, A., Tejero, J., Hermoso, J., Mayoral, T.,
Sanz-Aparicio, J., Grever, K. & Go
´
mez-Moreno, C. (2001)

Probing the determinants of coenzyme specificity in ferredoxin-
NADP
+
reductase by site-directed mutagenesis. J. Biol. Chem.
276, 11902–11912.
52. Martı
´
nez-Ju´ lvez, M., Nogue
´
s,I.,Faro,M.,Hurley,J.K.,Brodie,
T.B., Mayoral, T., Sanz-Aparicio, J., Hermoso, J., Stankovich,
M.T., Medina, M., Tollin, G. & Go
´
mez-Moreno, C. (2001) Role
of a cluster of hydrophobic residues near the FAD cofactor in
Anabaena PCC 7119 ferredoxin-NADP
+
reductase for optimal
complex formation and electron transfer to ferredoxin. J. Biol.
Chem. 276, 27498–27510.
53. Arakaki,A.K.,Orellano,E.G.,Calcaterra,N.B.,Ottado,J.&
Ceccarelli, E.A. (2001) Involvement of the flavin si-face tyrosine
on the structure and function of ferredoxin-NADP
+
reductases.
J. Biol. Chem. 276, 44419–44426.
54. Faro, M., Go
´
mez-Moreno,C.,Stankovich,M.&Medina,M.
(2002) Role of critical charged residues in reduction potential

modulation of ferredoxin-NADP
+
reductase. Eur. J. Biochem.
269, 2656–2661.
55. Faro, M., Frago, S., Mayoral,T.,Hermoso, J.A., Sanz-Aparicio, J.,
Go
´
mez-Moreno, C. & Medina, M. (2002) Probing the role of
glutamic acid 139 of Anabaena ferredoxin-NADP
+
reductase in
the interaction with substrates. Eur. J. Biochem. 269, 4938–4947.
56. Faro, M., Hurley, J.K., Medina, M., Tollin, G. & Go
´
mez-Mor-
eno, C. (2002) Flavin photochemistry in the analysis of electron
transfer reactions: role of charged and hydrophobic residues at the
carboxyl terminus of ferredoxin-NADP
+
reductase in the inter-
action with its substrates. Bioelectrochemistry 56, 19–21.
57. Hurley, J.K., Morales, R., Martı
´
nez-Ju´ lvez, M., Brodie, T.M.,
Medina, M., Go
´
mez-Moreno, C. & Tollin, G. (2002) Structure–
function relationships in Anabaena ferredoxin/ferredoxin:
NADP
+

reductase electron transfer: insights from site-directed
mutagenesis, transient absorption spectroscopy and X-ray crys-
tallography. Biochim. Biophys. Acta 1554, 5–21.
58. Ziegler, G.A. & Schulz, G.E. (2001) Crystal structures of adre-
nodoxin reductase in complex with NADP
+
and NADPH sug-
gesting a mechanism for the electron transfer of an enzyme family.
Biochemistry 39, 10986–10995.
59. Batie, C.J. & Kamin, H. (1984) Ferredoxin-NADP
+
oxidore-
ductase. Equilibria in binary and ternary complexes with NADP
+
and ferredoxin. J. Biol. Chem. 259, 8832–8839.
60. Batie, C.J. & Kamin, H. (1984) Electron transfer by ferredoxin-
NADP
+
reductase. Rapid-reaction evidence for participation of a
ternary complex. J. Biol. Chem. 259, 11976–11985.
61. Batie, C.J. & Kamin, H. (1986) Association of ferredoxin-
NADP
+
reductase with NADP(H). Specificity and oxidation-
reduction properties. J. Biol. Chem. 261, 11214–11223.
62. Leadbeater, C., McIver, L., Campoiano, D.J., Webster, S.P.,
Baxter, R.L., Kelly, S.M., Price, N.C., Lysek, D.A., Noble, M.A.,
Chapman, S.K. & Munro, A.W. (2000) Probing the NADPH
binding site of Escherichia coli flavodoxin oxidoreductase. Bio-
chem. J. 352, 257–266.

1914 N. Carrillo and E. A. Ceccarelli (Eur. J. Biochem. 270) Ó FEBS 2003
63. Wan, J.T. & Jarrett, J.T. (2002) Electron acceptor specificity of
ferredoxin (flavodoxin): NADP
+
oxidoreductase from Escheri-
chia coli. Arch. Biochem. Biophys. 406, 116–126.
64. Jarrett, J.T. & Wan, J.T. (2002) Thermal inactivation of reduced
ferredoxin (flavodoxin): NADP
+
oxidoreductase from Escheri-
chia coli. FEBS Lett. 529, 237–242.
65. Casaus, J.L., Navarro, J.A., Herva
´
s, M., Lostao, A., De la Rosa,
M.A., Go
´
mez-Moreno, C., Sancho, J. & Medina, M. (2002)
Anabaena sp. PCC 7119 flavodoxin as electron carrier from pho-
tosystem I to ferredoxin-NADP
+
reductase. Role of Trp(57) and
Tyr(94). J. Biol. Chem. 277, 22338–22344.
66. De Pascalis, A.R., Jelesarov, I., Ackermann, F., Koppenol, W.H.,
Hirasawa, M., Knaff, D.B. & Bosshard, H.R. (1993) Binding of
ferredoxin to ferredoxin-NADP
+
oxidoreductase: the role of
carboxyl groups, electrostatic surface potential, and molecular
dipole moment. Protein Sci. 2, 1126–1135.
67. Jelesarov, I., De Pascalis, A.R., Koppenol, W.H., Hirasawa, M.,

Knaff, D.B. & Bosshard, H.R. (1993) Ferredoxin binding site on
ferredoxin-NADP
+
reductase. Differential chemical modification
of free and ferredoxin-bound enzyme. Eur. J. Biochem. 216, 57–66.
68. Ullmann, G.M., Hauswald, M., Jensen, A. & Knapp, E.W. (2000)
Structural alignment of ferredoxin and flavodoxin based on
electrostatic potentials: implications for their interactions with
photosystem I and ferredoxin-NADP
+
reductase. Proteins 38,
301–309.
69. Morales, R., Charon, M.H., Kachalova, G., Serre, L., Medina,
M., Go
´
mez-Moreno, C. & Frey, M. (2000) A redox–dependent
interaction between two electron-transfer partners involved in
photosynthesis. EMBO Reports 1, 271–276.
70. Smith,J.M.,Smith,W.H.&Knaff,D.B.(1981)Electrochemical
titration of a ferredoxin-ferredoxin-NADP
+
reductase complex.
Biochim. Biophys. Acta 635, 405–411.
71. Bowler, C. & Chua, N.H. (1994) Emerging themes of plant signal
transduction. Plant Cell 6, 1529–1541.
72. Ritchie, S.W., Redinbaugh, M.G., Shiraishi, N., Vrba, J. &
Campbell, W.H. (1994) Identification of a maize root transcript
expressed in the primary response to nitrate: characterization of a
cDNA with homology to ferredoxin-NADP
+

oxidoreductase.
Plant Mol. Biol. 26, 679–690.
73. Onda, Y., Matsumura, T., Kimata-Ariga, Y., Sakakibara, H.,
Sugiyama, T. & Hase, T. (2000) Differential interaction of maize
root ferredoxin-NADP
+
oxidoreductase with photosynthetic and
non-photosynthetic ferredoxin isoproteins. Plant Physiol. 123,
1037–1045.
74. Isas, M.J. & Burgess, B.K. (1994) Purification and characteriza-
tion of a NADP
+
/NADPH-specific flavoprotein that is over-
expressedinFdI
–
strains of Azotobacter vinelandii. J. Biol. Chem.
269, 19404–19409.
75. Forti, G. & Sturani, E. (1968) On the structure and function of
reduced nicotinamide adenine dinucleotide phosphate-cyto-
chrome f reductase of spinach chloroplasts. Eur. J. Biochem. 3,
461–472.
76. Zanetti, G. & Forti, G. (1966) Studies on the triphosphopyridine
nucleotide-cytochrome f reductase of chloroplats. J. Biol. Chem.
241, 279–285.
77. Segel, I.W. (1975) Enzyme Kinetics. Wiley Interscience, New York,
USA.
78. Hajirezaei, M.R., Peisker, M., Tschiersch, H., Palatnik, J.F.,
Valle, E.M., Carrillo, N. & Sonnewald, U. (2002) Small changes in
the activity of chloroplast NADP
+

-dependent ferredoxin reduc-
tase lead to impaired plant growth and restrict photosynthetic
capacity of transgenic tobacco plants. Plant J. 29, 281–293.
79. Fersht, A. (1999) Structure and Mechanism in Protein Science.
Freeman, New York, USA.
80. Albery, W.J. & Knowles, J.R. (1976) Evolution of enzyme func-
tion and the development of catalytic efficiency. Biochemistry 15,
5631–5640.
81. Penfound, T. & Foster, J.W. (1996) Biosynthesis and recycling of
NAD. In Escherichia coli and Salmonella Cellular and Molecular
Biology (Neidhart, F.C., ed.), pp. 721–730. ASM Press, Wash-
ington, DC, USA.
Ó FEBS 2003 Ferredoxin-NADP(H) reductase catalytic mechanism (Eur. J. Biochem. 270) 1915