Báo cáo khoa học: Dynamics of flavin semiquinone protolysis in L-a-hydroxyacid-oxidizing flavoenzymes – a study using nanosecond laser flash photolysis doc

Dynamics of flavin semiquinone protolysis in
L-a-hydroxyacid-oxidizing flavoenzymes – a study
using nanosecond laser flash photolysis
Lars Lindqvist
1
, Simona Apostol
1,
*, Chaibia El Hanine-Lmoumene
1
and Florence Lederer
2
1 Laboratoire de Photophysique Mole
´
culaire du Centre National de la Recherche Scientifique, Universite
´
Paris-Sud, 91405 Orsay, France
2 Laboratoire de Chimie Physique, Centre National de la Recherche Scientifique UMR 8000, Universite
´
Paris-Sud, 91405 Orsay, France
Introduction
Proton transfer is involved in most if not all biological
processes, as well as in formation of the structures
of biological macromolecules. In soluble enzymes,
acid–base catalysis is of fundamental importance. In
biomembranes, electron and proton transfers are often
coupled. Determining the kinetics of proton transfers
in an individual system is of prime importance in
understanding the fine details of the processes involved
at the functional and structural levels. However, the
dynamics of proton movements are not always easy to
analyze for biological phenomena, either because of

the lack of a directly observable signal, or because
Keywords
flavin semiquinone; flavocytochrome b
2
;
long-chain hydroxy acid oxidase;
nanosecond laser photolysis; proton transfer
kinetics
Correspondence
L. Lindqvist, Laboratoire de Photophysique
Mole
´
culaire du CNRS, Universite
´
Paris-Sud,
91405 Orsay Cedex, France
Fax: +33 1 69156777
Tel: +33 1 69157909
E-mail:
*Present address
Physics Department, Faculty of Sciences
and Arts, Targoviste, Romania
(Received 10 October 2009, revised 2
December 2009, accepted 7 December
2009)
doi:10.1111/j.1742-4658.2009.07539.x
The reactions of the flavin semiquinone generated by laser-induced stepwise
two-photon excitation of reduced flavin have been studied previously
(El Hanine-Lmoumene C & Lindqvist L. (1997) Photochem Photobiol 66,
591–595) using time-resolved spectroscopy. In the present work, we have

used the same experimental procedure to study the flavin semiquinone in
rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase
domain of flavocytochrome b
2
FDH, two homologous flavoproteins
belonging to the family of FMN-dependent
L-2-hydroxy acid-oxidizing
enzymes. For both proteins, pulsed laser irradiation at 355 nm of the
reduced enzyme generated initially the neutral semiquinone, which has
rarely been observed previously for these enzymes, and hydrated electron.
The radical evolved with time to the anionic semiquinone that is known to
be stabilized by these enzymes at physiological pH. The deprotonation
kinetics were biphasic, with durations of 1–5 ls and tens of microseconds,
respectively. The fast phase rate increased with pH and Tris buffer concen-
tration. However, this increase was about 10-fold less pronounced than
that reported for the neutral semiquinone free in aqueous solution. pK
a
values close to that of the free flavin semiquinone were obtained from the
transient protolytic equilibrium at the end of the fast phase. The second
slow deprotonation phase may reflect a conformational relaxation in the
flavoprotein, from the fully reduced to the semiquinone state. The anionic
semiquinone is known to be an intermediate in the flavocytochrome b
2
catalytic cycle. In light of published kinetic studies, our results indicate that
deprotonation of the flavin radical is not rate-limiting for the intramolecu-
lar electron transfer processes in this protein.
Abbreviations
FDH, flavodehydrogenase domain of flavocytochrome b
2
; FMN

•)
, anionic FMN semiquinone; FMNH
)
, fully reduced FMN anion; FMNH
•
,
neutral FMN semiquinone; LCHAO, recombinant long-chain hydroxy acid oxidase from rat.
964 FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS
another process is rate-limiting. The most successful
method for studies in biological systems has been
laser-induced pH jump [1], where a transient pH
change is obtained by proton ejection from a photodis-
sociable dye. Time-resolved studies of proton transfer
for a number of proteins using this method have been
reviewed by Gutman and Nachliel [2] and A
ă
delroth
and Brzezinski [3]. However, the method is limited to
a narrow time window because of rapid pH relaxation
to the initial state after the pH jump.
We have instead made use of the transformation of
a photoreactive species into a stable product in this
case the FMN semiquinone, obtained from the fully
reduced FMN anion (FMNH
)
) thus allowing a
time-resolved study of protolytic reactions involving
the avin radical without limitation of the observation
time. The photochemical reaction was achieved by
two-photon excitation of the reduced avin. Indeed,

our previous studies [4,5] of FMNH
)
in aqueous solu-
tion showed that pulsed laser excitation of the reduced
avin at 355 nm gives rise to one-electron ionization at
high laser intensities by stepwise two-photon absorp-
tion, with formation of the hydrated electron (e
aq
)
)
and the neutral FMN semiquinone (FMNH

):
FMNH

ỵ 2hv ! FMNH

ỵ e

aq
1ị
In those experiments, the avin radical appeared
initially as the neutral (blue) species (FMNH

)atpH
7-10; however, acidbase equilibrium of the radical was
attained within a few microseconds by deprotonation
of a proportion of the neutral radical to the anionic
(red) form (FMN
)

). This laser-induced reaction pro-
vides an exclusive means of studying protolysis dynam-
ics in avoenzymes. In this paper, we report results
obtained for two members of a avoenzyme family that
oxidizes l-2-hydroxy acids: rat long-chain hydroxy acid
oxidase (LCHAO, EC 1.1.3.15, isozyme B) and the
avodehydrogenase domain (FDH) of avocytochrome
b
2
, a lactate dehydrogenase from yeast (EC 1.1.2.3).
These proteins have been well characterized at the func-
tional and structural level [6,7]. Their crystal structures
show a high degree of similarity, both in the b
8
a
8
fold
and around the avin [810]. Family members stabilize
the anionic semiquinone at physiological pH [1115].
This has been demonstrated to be the case for avo-
cytochrome b
2
and its FDH domain [11,12] and should
be the case for LCHAO, which is an isozyme of spin-
ach glycolate oxidase [6,16]. The semiquinone pK
a
has
been determined only for lactate oxidase from
Aerococcus viridans, and was found to be 6.0 [14]. Part
of the present results have been presented previously in

preliminary form [17].
Results
Photoionization reaction
The avoprotein solutions in the relevant buffer (see
Fig. 1 for details) ushed with argon were exposed to
laser pulses of varying uence. The appearance of e
aq
)
was measured at the 715 nm absorption peak of this
species [18] at the end of the laser pulse, and the e
aq
)
concentration was calculated using the extinction coef-
cient 1.85 ã 10
4
m
)1
ặcm
)1
[18]. The results (Fig. 1)
revealed that e
aq
)
is formed for both proteins, conrm-
ing the occurrence of photoionization (Eqn 1). The
e
aq
)
then disappeared within about one microsecond.
A previous study of the FDH domain by transient

absorption spectroscopy at sub-picosecond time resolu-
tion [19] showed that the excited singlet state of the
fully reduced avin has a lifetime long enough in this
protein (approximately 1.5 ns) to be populated to a
large extent by the laser pulse and to absorb a second
photon at the uence rates used here.
As photoionization is a two-photon process, one
would expect the e
aq
)
yield to increase quadratically
with the laser uence. However, a previous study of
free FMNH
)
in aqueous solution [5] showed that the
formation of e
aq
)
was proportional to the square of
the laser uence only at the lowest uences, and then
increased quasi-linearly with the uence. The deviation
from a quadratic response was ascribed to depletion of
ground-state avin during the laser pulse, concurrent
with screening effects caused by absorption of the laser
light by transient species. The present results show the
same behaviour: formation of e
aq
)
was noticeable only
above a certain uence threshold and then increased

almost linearly with the uence.
5
10
15
LCHAO
e
aq

e
aq

FMNH
ã
0 0.02 0.04 0.06
0
0 0.02 0.04
0
2
4
6
Laser fluence (J cm
2
)
FDH
FMNH
ã
Transient conc. (à
M
)
Fig. 1. Yields of e

aq
)
and FMNH

obtained upon laser excitation at
the end of the laser pulse for the FDH domain (70 l
M in 25 mM
Tris H
2
SO
4
, pH 7.8) and LCHAO (150 lM in 10 mM Tris HCl,
pH 7.5).
L. Lindqvist et al. Flavin semiquinone protolysis
FEBS Journal 277 (2010) 964972 ê 2010 The Authors Journal compilation ê 2010 FEBS 965
The formation of FMNH
•
at the end of the laser
pulse was measured at 570 nm in N
2
O-saturated
solutions to scavenge e
aq
)
. FMNH
•
concentrations
were obtained using the extinction coefficient
5 · 10
3

m
)1
Æcm
)1
reported for FMNH
•
obtained from
free flavin and several flavoproteins at the absorption
maximum in the visible spectrum [20,21]. Figure 1
shows that the formation of FMNH
•
, as found for
e
aq
)
, increases almost linearly with the laser fluence;
however, the FMNH
•
concentration is higher than that
of e
aq
)
, in contrast to the stoichiometric formation
expected from Eqn (1). The reasons for this discrep-
ancy were investigated by comparing these results with
those obtained in parallel with free FMNH
)
under the
same conditions. These experiments showed that
FMNH

•
is formed in equal amounts (within ±10%)
in both cases, assuming that the extinction coefficients
of the flavin radical have the same values in free and
protein-bound conditions. This finding strongly sug-
gests that the photoionization efficiency is the same for
the FDH domain and for free FMNH
)
. However,
comparison of the e
aq
)
yields gave a considerably
lower value, approximately 60%, for the protein com-
pared to that for free FMNH
)
. The deficit in e
aq
)
yield
for the two proteins may be explained by assuming
that part of the e
aq
)
just released by laser excitation
reacts in sub-nanosecond time with amino acid resi-
dues in the protein during its diffusion from the flavin
towards the surrounding aqueous solution, and thus
escapes observation. It is interesting to note that a pre-
vious study of Desulfovibrio vulgaris flavodoxin [22]

showed that e
aq
)
and the flavin radical are formed in
stoichiometric ratio, as expected from Eqn (1). The fla-
vin environment in flavodoxin is very different from
that of the two l-2-hydroxy acid dehydrogenases. In
flavodoxin, a tyrosine residue protects part of the fla-
vin si face from the solvent, but the benzenoid ring
methyl groups are exposed [23]. In the two homolo-
gous enzymes studied here, N5 is practically the only
FMN atom accessible to the solvent, in a shallow
active site, which may be occluded some of the time by
a mobile loop that is partly invisible in the crystal
structures of the two enzymes [8–10]. How these struc-
tural differences compared with flavodoxin affect the
fate of e
aq
)
is unclear.
Flavin semiquinone spectra
The absorption spectra of the half-reduced flavin
formed upon laser excitation of the FDH domain and
of LCHAO, in 50 mm Tris buffer, pH 7.5-7.8, were
determined by measuring the transient absorbance
changes between 320 and 670 nm at the end of the
laser pulse in N
2
O-saturated solutions. The difference
spectra thus obtained were extrapolated to 100% con-

version of the flavin to FMNH
•
(the laser pulse
achieved up to 10% conversion) using the extinction
coefficient 5 · 10
3
m
)1
Æcm
)1
at 570 nm. Addition of
these difference spectra to the absorption spectra of
the reduced flavoproteins gave the spectra shown in
Fig. 2, which are characteristic of neutral flavin radi-
cals and compare well with spectra reported by others
[20,21,24].
The ‘end-of-pulse’ spectra evolved within about
0.1 ms into spectra characteristic of the flavin anion
radical as shown in Fig. 2. It is known that the semi-
quinone is anionic in the neutral pH range in the pres-
ent flavoproteins [11,12,15]; therefore, the initially
generated neutral radicals are expected to undergo
deprotonation to yield the anionic radical in the pH
range studied (7.5–9.7).
FMNH
•
deprotonation kinetics
The deprotonation of FMNH
•
after the end of the laser

pulse was studied in the wavelength range 360-600 nm
at various pH values. Figure 3 shows individual curves
illustrating the kinetics at 570 nm, where FMNH
•
is the
only species absorbing significantly. It can be seen that
LCHAO
300 400 500 600 700300 400 500 600
0
4000
8000
12000
16000
Wavelen
g
th (nm)
Extinction coefficient (M
–1
·cm
–1
)
FDH
Fig. 2. Absorption spectra of the species formed upon laser excita-
tion of the FDH domain and of LCHAO at pH 7.5–7.8 (50 m
M Tris
buffer) in N
2
O-saturated solutions. The transient absorbance
changes obtained on laser excitation were extrapolated to 100%
conversion of the flavin to FMNH

•
(the laser pulse achieved up to
10% conversion) and added to the absorption spectra of the
respective reduced flavoproteins. Open triangles, FMNH
•
obtained
at the end of the laser pulse; closed circles, FMN
•)
obtained at the
end of the ‘slow’ phase; full line, absorption spectra of the fully
reduced proteins.
Flavin semiquinone protolysis L. Lindqvist et al.
966 FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS
evolution of the transient absorbance is complex,
comprising a ‘fast’ phase lasting 1–5 ls and a ‘slow’
phase lasting up to tens of microseconds. The kinetics
could be expressed satisfactorily by bi-exponentials with
rate parameters independent of wavelength. At the
end of the ‘slow’ phase, the absorbance was found to
correspond mainly to that of FMN
•)
(Fig. 2) in the pH
range studied. The amplitude of the ‘fast’ phase, deter-
mined by the disappearance of FMNH
•
, was found to
increase with pH at the expense of that of the ‘slow’
phase, as seen in Fig. 3.
The findings are illustrated in Fig. 4, in which the
absorbance remaining at 570 nm at the end of the

‘fast’ phase (DA
end
, after 1–5 ls), normalized with
respect to the absorbance variation at the end of the
laser pulse (DA
0
), is plotted against pH. If one assumes
that the ‘fast’ phase leads to a ‘temporary’ protolytic
equilibrium of the newly generated neutral radical with
the external solution, one can derive the pK
a
of the
radical at this stage. The smooth curves in the figure
represent the calculated fractional absorbance of the
neutral flavin radical, fitted to the experimental values
by setting pK
a
= 8.1 for the FDH domain and 8.7 for
LCHAO. It is striking that these values are close to
those (8.3–8.7) determined by diverse methods for the
neutral flavin radical free in aqueous solution [4,20,25–
28]. Indeed, one would instead expect a semiquinone
pK
a
value below 7 for these proteins, as mentioned
above [12,14,16], and therefore complete deprotona-
tion. However, the neutral radical still present at the
‘temporary’ equilibrium undergoes close to complete
deprotonation at the rate of the ‘slow’ phase, in agree-
ment with the result expected from the literature.

The rate constants of the ‘fast’ and ‘slow’ phases (k
1
and k
2
, respectively) reveal trends in the pH depen-
dence of the deprotonation rates (Table 1). For the
FDH domain, k
1
increases from pH 7.7 to 8.5. For
LCHAO, the ‘fast’ phase is absent at pH 7.5, reflecting
the higher pK
a
value of the flavin radical in this pro-
tein at its ‘temporary’ protolytic equilibrium compared
to that in the FDH domain (Fig. 4). However, the
‘fast’ phase appears at higher pH and its rate increases
with pH, as for the FDH domain.
7.0 7.5 8.0 8.5 9.0 9.5
0.0
0.2
0.4
0.6
0.8
1.0
LCHAO
pK
a
= 8.7
FDH
pK

a
= 8.1
ΔA
end
/ΔA
0
pH
Fig. 4. Titration curves for the equilibrium FMNH
•
⁄ FMN
•)
in the
FDH domain (+) and for LCHAO (open circles) in 50 m
M Tris buffer,
obtained at the end of the ‘fast’ deprotonation phase of FMNH
•
.
The ratio of the absorbance variation at 570 nm at the end of this
phase (DA
end
) to that at the moment of the laser pulse (DA
0
)is
plotted against pH. The smooth curves were obtained from the
expression for DA
end
⁄ DA
0
expected at protolytic equilibrium:
DA

end
⁄ DA
0
=1) (1 ) r) ⁄ (10
(pK ) pH)
+ 1), where r is the residual
absorbance due to the ‘slow’ phase. The calculated curves were
fitted to the experimental values by setting pK
a
to 8.1 (r = 0.03) for
the FDH domain and to 8.7 (r = 0.2) for LCHAO.
0.00
0.02
0.04
pH 9.6
pH 8.45
LCHAO
pH 7.5
Time (µs)
0102030
020406080
0.00
0.01
0.02
0.03
FDH
pH 8.3
pH 7.7
ΔAbsorbance
Time (µs)

Fig. 3. Time evolution of the flavin radical in the FDH domain and
in LCHAO, measured after the end of the laser pulse at various pH
values, in 50 m
M Tris buffer, measured at 570 nm. The smooth
lines are bi-exponential fits to the curves, using a computer pro-
gram based on the Levenberg–Marquardt non-linear least-squares
fit algorithm. The curves show individual measurements but are
representative of a number of experiments.
Table 1. Rate parameters of FMNH
•
deprotonation. Rate constants
of FMNH
•
deprotonation in the FDH domain and in LCHAO in
50 m
M Tris buffer at various pH values, obtained from measure-
ments of transient absorption decays at 570 nm. The smooth lines
are bi-exponential fits to the curves (see Fig. 3). The parameters
are mean values obtained from several independent experiments.
pH k
1
(· 10
6
s
)1
) k
2
(· 10
4
s

)1
)
FDH 7.7 0.23 ± 0.05 3 ± 1
8.0 0.43 ± 0.06 3 ± 1
8.3 0.53 ± 0.06 3 ± 1
8.5 0.60 ± 0.06 3 ± 1
LCHAO 7.5 0 2 ± 1
8.25 0.15 ± 0.05 2 ± 1
8.7 0.4 ± 0.1 7 ± 2
9.6 1.2 ± 0.3 16 ± 4
L. Lindqvist et al. Flavin semiquinone protolysis
FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS 967
As mentioned above, the neutral radical still present
at the end of the ‘fast’ phase undergoes deprotonation
at a slower rate to yield the protolytically stable radi-
cal in these proteins. It can be seen from Table 1 that
the rate constant (k
2
) of the ‘slow’ phase is approxi-
mately the same for the two proteins (2–3 · 10
4
s
)1
)at
the lowest pH (pH 7.5-7.7), even when the ‘fast’ phase
is absent. At higher pH, the k
2
value appears to
increase with pH for LCHAO.
The deprotonation may occur by transfer from the

radical to the various proton acceptors present in the
solutions (Tris, EDTA, H
2
O and OH
)
) and ⁄ or to pro-
tein side chains. Control runs at 2 and 5 mm EDTA
gave the same deprotonation rates. Thus, a reaction
with EDTA can be neglected, and so can a reaction
with OH
)
at the pH values used here. This leaves Tris
and H
2
O as possible proton acceptors:
FMNH

þ Tris ! FMN
À
þ TrisH
þ
ð2Þ
FMNH

þ H
2
O ! FMN
À
þ H
3

O
þ
ð3Þ
The contribution of Tris buffer to the deprotonation
was determined by studying the effect of its concentra-
tion on the ‘fast’ deprotonation rates for the FDH
domain. Figure 5A shows examples of transient absor-
bance curves obtained at 570 nm in 10 and 100 mm
Tris buffer (pH 7.7). It can be seen that the deprotona-
tion rate increases with increase in buffer concentra-
tion. The rate constant of the ‘fast’ deprotonation
process, obtained from the transient absorbance at
570 nm, is shown in Fig. 5B as a function of the Tris
concentration. The rate constant increases linearly with
the buffer concentration, and can be expressed as k
1
=
k
0
+ k
Tris
· [Tris], where k
Tris
is approximately
1.4 · 10
6
m
)1
Æs
)1

. This value may be confronted with
results obtained previously for free FMNH
•
in aque-
ous solution [22]. The deprotonation rate constant of
the neutral flavin radical by Tris at pH 8.7 was deter-
mined to be 2.2 · 10
7
m
)1
Æs
)1
, i.e. one order of magni-
tude faster in aqueous solution than in the protein
environment. This finding is in line with the idea that
when partially embedded in the protein, the flavin is
less accessible to the buffer than when it is fully
exposed to the aqueous solution. Figure 5B shows that
the deprotonation is fast even in the absence of buffer,
with a rate constant k
0
of 1.4 · 10
5
s
)1
.
Discussion
The two homologous enzymes analyzed in this study
exhibit essentially the same behavior on exposure to
laser flash excitation at 355 nm: at the end of the laser

pulse, one electron has been ejected from the flavin,
which is then in the neutral semiquinone state. The
pK
a
of this species was found to be close to that of
free flavin (Fig. 4). This neutral radical then undergoes
deprotonation to yield the stable anionic semiquinone,
previously characterized by spectrophotometry [12]
and EPR [11]. Hazzard et al. [29] proposed that a neu-
tral semiquinone is formed in flavocytochrome b
2
by
electron transfer from FMNH
)
to heme b
2
upon back
reduction of the heme after partial enzyme oxidation
by the laser-generated triplet state of 5-deazariboflavin.
However, these authors did not study the fate over
time of this neutral semiquinone.
In the present study, the FMNH
•
deprotonation
appears to be biphasic for both proteins. In a first
rapid phase, the radical protonation state adjusts to
the buffer pH as if the semiquinone were free in solu-
tion. For the fast deprotonation phase of the FDH
domain at pH 7.7, the Tris-dependent and Tris-inde-
pendent rate constants were approximately 1.4 · 10

6
m
)1
Æs
)1
and 1.4 · 10
5
s
)1
, respectively (Fig. 5).
The conclusion must then be that it is water itself
that is responsible for the Tris-independent deprotona-
tion of the neutral semiquinone, according to Eqn (3).
However, this idea raises a problem. With H
2
O as the
proton acceptor and a pK
a
of )1.74 for the hydronium
ion, one would expect the Tris-independent rate to be
at least two orders of magnitude lower. Furthermore,
the value of the Tris-dependent rate constant is insuffi-
cient to explain the variation of the fast phase rate
with pH if the Tris-independent rate is constant, as
expected for an exchange with water in the explored
pH range. A tentative answer to the problem may be
0
0.01
0.02
0.03

0.04
0.05
A
B
ΔAbsorbance
10 m
M
Tris
100 m
M
Tris
Time (µs)
0 20 40 60 80 0.00 0.03 0.06 0.09
0.0
0.1
0.2
0.3
k
1
x 10
–6
(s
–1
)
[Tris] (M)
Fig. 5. Influence of buffer concentration on the kinetics of the fla-
vin radical evolution. (A) Absorbance variation at 570 nm obtained
upon laser excitation of 70 l
M FDH domain solutions (saturated
with N

2
O) buffered at pH 7.7 with 10 or 100 mM Tris. Smooth
curves are bi-exponential fits to the experimental curves. (B) Rate
constant (k
1
) of the ‘fast’ phase describing deprotonation of the
neutral flavin radical measured at 570 nm, plotted against Tris con-
centration. The linear fit gives k
1
= (1.4 · 10
5
+ 1.4 · 10
6
· [Tris])
s
)1
.
Flavin semiquinone protolysis L. Lindqvist et al.
968 FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS
obtained by considering that the reaction between
FMNH
•
and water does not take place in bulk water
but at the surface of the protein. Inspection of the
FDH domain crystal structure [9,10] appears to rule
out the possibility that amino acid side chains in the
vicinity of the flavin could take part directly in the
deprotonation, as these chains (two tyrosines and a
histidine) are too distant and ⁄ or in a wrong orienta-
tion for direct hydrogen bonding to the N5 hydrogen

of FMNH
•
as required for proton abstraction. Never-
theless, the active site of these enzymes on the flavin si
side is highly polar, with two arginines and ionizable
side chains such as a histidine and two tyrosines in the
FDH domain, with one of the latter being replaced in
LCHAO by a phenylalanine. The pK
a
of the tyrosines
is not known, but it has been determined for flavocyto-
chrome b
2
that the active site histidine (H373) has an
abnormal pK
a
of 9.1 in the reduced enzyme [30]. The
crystal structure suggests that the pK
a
of this residue is
still elevated when the flavin is in the semiquinone
state [10]. It is thus not impossible that electrostatic
factors, together with interaction of the active-site resi-
dues with specific water molecules via hydrogen bond-
ing, may accelerate the Tris-independent deprotonation
compared to what is expected in bulk water. The
variation of the electrostatic environment with pH may
then contribute to the variation of the fast phase rate
with pH. In addition, a role may also be played by
residues in mobile loop 4 of the b barrel, which are

partly invisible in the crystal structures of both
proteins [8–10]. The boundaries of the invisible region
lie 15–20 A
˚
away from the flavin, but there is experi-
mental evidence that modifications in this loop have
an impact on the flavin environment [6,31].
In the second ‘slow’ phase, the pK
a
of the radical
shifts to a value below 7 as expected from the litera-
ture. The origin of the slow phase is intriguing. We
propose that evolution of the flavin radical pK
a
in
these enzymes is due to protein conformational
changes upon passing from the fully reduced state to
the half-reduced state. At the time of laser-induced
generation of the flavin radical, the protein is present
in its conformation in the fully reduced state, but then
undergoes relaxation to the conformation in the half-
reduced state. The high pK
a
values associated with the
fast deprotonation phase would then correspond to the
pK
a
of the half-reduced flavin before conformational
relaxation. A lower pK
a

prevails in the conformation-
ally relaxed protein, thus explaining the transition to
the anionic semiquinone. On the basis of this hypothe-
sis, the deprotonation rate of the slow phase would be
the rate of the conformational change(s). Yet, at pres-
ent, there is no experimental evidence in support of
this hypothesis. The crystal structure of the FDH
domain in holo-flavocytochrome b
2
is known at 2.3 A
˚
resolution [9,10]. At that resolution, the structure
shows no significant difference between the fully
reduced subunit and the subunit with flavin in the
semiquinone state complexed with the product pyru-
vate. In both subunits, the flavin ring is slightly bent,
and the rmsd for atomic positions of the FMN groups
is 0.17 A
˚
, with most of the deviations being localized
in the phosphate regions [9]. Structures at atomic
resolution are required in order to see whether the
structural adjustment between the two redox states is
due to a difference in the flavin planarity. For
LCHAO, only the structure of the oxidized enzyme is
known [8].
LCHAO is an oxidase; the fully reduced flavin is
re-oxidized at the expense of oxygen, with formation of
hydrogen peroxide. Although this enzyme stabilizes the
anionic semiquinone, the latter has never been observed

as an intermediate in the catalytic cycle, no more than in
most other flavo-oxidases [32,33]. In contrast, in the
flavocytochrome b
2
catalytic cycle, the semiquinone is
an EPR-detectable intermediate [11]. After being
reduced by the substrate in a two-electron reaction, the
flavin yields electrons one at a time to the heme in the
same subunit [7]. NMR studies at neutral pH demon-
strated that, in the reduced enzyme, the flavin is proton-
ated at N5 [34]; therefore it must lose the proton in
order to form the anionic semiquinone after the first
electron is transferred to the heme. Values for the rate of
FMN
•)
formation from FMNH
)
(equal to the rate of
heme reduction by FMNH
)
) have been calculated from
stopped-flow experiments under various experimental
conditions [11,35–37]. In 10 mm Tris ⁄ HCl, with I = 0.1
(25 °C), conditions close to those used here, the heme
reduction rate was estimated to be of the order of
1.5 · 10
3
s
)1
[35,38]. Thus, the rate determined in this

work for the slow event leading to deprotonation of the
neutral radical is about 10-fold faster than the electron
transfer rate estimated in independent kinetic studies.
The loss of the N5 proton initially present in FMNH
)
is
therefore not rate-limiting for heme reduction.
In conclusion, the study shows that the experimental
method proposed here makes possible the investigation
of protolytic reactions in flavoproteins at high tempo-
ral resolution. The results reveal an unexpected
complexity in the kinetics of these reactions for the
two enzymes studied, attributed hypothetically to a
conformational relaxation induced by the change in
the flavin redox state. However, extension of the study
to other flavoenzyme classes and other buffers as well
as additional structural information are required to
substantiate this hypothesis.
L. Lindqvist et al. Flavin semiquinone protolysis
FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS 969
Experimental procedures
Laser flash photolysis
The laser flash photolysis set-up has been described previ-
ously [5,39]. The third harmonic (k = 355 nm) obtained
from a pulsed (approximately 2 ns full width at half maxi-
mum) Nd ⁄ YAG laser was used for photoexcitation and a
pulsed Xe UV lamp was used as the probing light source,
in crossed-beam configuration. Samples under study were
contained in 1 · 1 cm silica cuvettes with polished windows
and equipped with glass tubing for degassing. The laser

beam was shaped to 1 cm width and 0.3 cm height at the
laser entrance window. A ground silica plate in front of the
window ensured homogeneous irradiation. A diaphragm at
the probe beam entrance window defined a 0.2 · 0.3 cm
beam (width · height) passing through the 1 cm cuvette
path adjacent to the laser entrance window. The intensity
of the transmitted probe light was measured at selected
wavelengths as a function of time using a monochroma-
tor ⁄ photomultiplier ⁄ digital oscilloscope device.
The fluence of the laser pulses at the entrance window of
the sample cuvette was determined by ‘anthracene triplet
actinometry’ [40]. In this case, the laser intensity was atten-
uated using calibrated neutral filters to avoid saturation
effects.
The reduced flavoproteins are weakly fluorescent. The
fluorescence emitted during the laser pulse interfered with
the measurement using the Xe lamp, and the measurements
during and immediately after the laser pulse (up to 5 ls)
were therefore performed using this lamp at a light intensity
that was high enough to make the perturbation by the fluo-
rescence pulse acceptable. The intensity of the probe light
remained constant within 1% over 5 ls under this regime.
For measurements at longer times, the Xe lamp was used
at a lower intensity. Under this regime, the probe light
remained constant to within 0.5% over 5 ls after laser exci-
tation (absorbance error ±0.001) and within 2% over
100 ls (absorbance error ±0.004).
Protein preparations
Recombinant LCHAO was prepared as described previously
[6], except that DEAE Sepharose Fast Flow (Pharmacia,

Orsay, France) was used instead of DEAE cellulose for the
second chromatographic step. Samples (0.1–0.3 mm) were
prepared for laser study after dialysis against Tris ⁄ HCl,
10 mm EDTA. HCl ⁄ NaOH was used to set the pH.
The recombinant FDH domain was prepared as described
previously [12]. For the laser flash experiments, 0.06-0.12 mm
solutions (in terms of flavin) were prepared after dialysis
against Tris ⁄ H
2
SO
4
,5mm EDTA. H
2
SO
4
⁄ NaOH was used
to set the pH, and K
2
SO
4
to adjust the ionic strength, as the
chloride ion has been reported to inhibit the enzyme by
binding to the active site [12].
The flavoprotein solutions (3 mL) were de-aerated in the
cuvettes by flushing N
2
O (argon in the study of e
aq
)
) above

the solution surface over 1 h on ice with gentle rocking.
The cuvettes were then closed by means of a septum, and
the solutions were exposed at 23 ° C to the visible light from
a DC Xe lamp, ensuring photoreduction of the flavin by
the EDTA present in the solutions. In the case of the FDH
domain, it was necessary to reduce the major part of the
flavin by adding 200 lLof30mm lithium l-lactate before
photoreduction. The final l-lactate concentration (1.9 mm)
was far below the concentration that inhibits the enzyme by
binding to the reduced form (several hundred millimolar
[41]). Similarly, the amount of pyruvate formed (in princi-
ple no more than the enzyme concentration) should have
been low compared to that required for binding to the
reduced or semiquinone forms [12,42]. Therefore, this pro-
cedure did not introduce a bias in the comparison with
LCHAO.
Exposure to the UV laser pulses resulted in oxidation of
the reduced flavoproteins to a slight extent. The solutions
were therefore regularly exposed to visible light to re-reduce
oxidized flavin. However, the polypeptide chains were also
gradually destroyed as shown by activity tests, and the
solutions were discarded after a few tens of laser shots
(5–10% activity loss). The results were not affected by
degradation within this limit.
At the enzyme concentrations used in this work, sponta-
neous flavin dissociation could not have taken place.
Indeed, for flavocytochrome b
2
purified from yeast, the
flavin K

d
value was in the 10
)8
to 10
)10
m range, depending
on the observation conditions [7]. No values have been
determined for the recombinant FDH domain or LCHAO,
but these proteins appeared as stable in this respect during
handling as flavocytochrome b
2
.
References
1 Smith KK, Kaufmann KJ, Huppert D & Gutman M
(1979) Picosecond proton ejection: an ultrafast pH
jump. Chem Phys Lett 64, 522–527.
2 Gutman M & Nachliel E (1997) Time-resolved
dynamics of proton transfer in proteinaceous systems.
Annu Rev Phys Chem 48, 329–356.
3A
¨
delroth P & Brzezinski P (2004) Surface-mediated
proton-transfer reactions in membrane-bound proteins.
Biochim Biophys Acta 1655, 102–115.
4 El Hanine-Lmoumene C & Lindqvist L (1997) Stepwise
two-photon excitation of 1,5-dihydroflavin mononucleo-
tide. Study of flavosemiquinone properties. Photochem
Photobiol 66, 591–595.
5 Lindqvist L (1993) Two-photon ionization of dihydro-
flavin mononucleotide on nanosecond laser excitation at

355 nm. J Photochem Photobiol B Biol 17, 27–33.
6 Belmouden A & Lederer F (1996) The role of a b barrel
loop 4 extension in modulating the physical and
Flavin semiquinone protolysis L. Lindqvist et al.
970 FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS
functional properties of long-chain 2-hydroxy-acid
oxidase isozymes. Eur J Biochem 238, 790–798.
7 Lederer F (1991) Flavocytochrome b
2
.InChemistry and
Biochemistry of Flavoenzymes (Mu
¨
ller F ed), pp. 153–
242. CRC Press, Boca Raton, FL.
8 Cunane LM, Barton JD, Chen Z-w, Leˆ KHD, Amar D,
Lederer F & Mathews FS (2005) Crystal structure anal-
ysis of recombinant rat kidney long-chain hydroxy acid
oxidase. Biochemistry 44, 1521–1531.
9 Cunane LM, Barton JD, Chen ZW, Welsh FE,
Chapman SK, Reid GA & Mathews FS (2002)
Crystallographic study of the recombinant flavin-
binding domain of baker’s yeast flavocytochrome b
2
:
comparison with the intact wild-type enzyme.
Biochemistry 41, 4264–4272.
10 Xia ZX & Mathews FS (1990) Molecular structure of
flavocytochrome b
2
at 2.4 A

˚
resolution. J Mol Biol 212,
837–863.
11 Capeille
`
re-Blandin C, Bray RC, Iwatsubo M &
Labeyrie F (1975) Flavocytochrome b
2
: kinetic studies
by absorbance and electron-paramagnetic-resonance
spectroscopy of electron distribution among prosthetic
groups. Eur J Biochem 54, 549–566.
12 Ce
´
nas N, Leˆ KHD, Terrier M & Lederer F (2007)
Potentiometric and further kinetic characterization of
the flavin-binding domain of Saccharomyces
cerevisiae flavocytochrome b
2
. Inhibition by anion
binding in the the active site. Biochemistry 46, 4661–
4670.
13 Ghisla S & Massey V (1991) l-lactate oxidase. In
Chemistry and Biochemistry of Flavoenzyme (Mu
¨
ller F
ed), pp. 243–249. CRC Press, Boca Raton, FL.
14 Maeda-Yorita K, Aki K, Sagai H, Misaki H & Massey
V (1995) l-lactate oxidase and l-lactate monooxygen-
ase: mechanistic variations on a common structural

theme. Biochimie 77, 631–642.
15 Massey V, Mu
¨
ller F, Feldberg R, Schuman M, Sullivan
PA, Howell LG, Mayhew SG, Matthews RG & Foust
GP (1969) The reactivity of flavoproteins with sulfite.
Possible relevance to the problem of oxygen reactivity.
J Biol Chem 244, 3999–4006.
16 Pace C & Stankovich M (1986) Oxidation–reduction
properties of glycolate oxidase. Biochemistry 25,
2516–2522.
17 El Hanine-Lmoumene C, Lindqvist L, Lederer F &
Serbanescu R (1997) Fast evolution of the pK
a
of
protein-bound flavin semiquinone generated
photochemically using UV laser irradiation. In Flavins
and Flavoproteins 1996 (Stevenson K, Massey V &
Williams CH Jr eds), pp. 139–142. University of Calgary
Press, Calgary, Canada.
18 Hart EJ & Anbar M (1970) The Hydrated Electron.
Wiley Interscience, New York.
19 Enescu M, Lindqvist L & Soep B (1998) Excited state
dynamics of fully reduced flavins and flavoenzymes,
studied at subpicosecond time resolution. Photochem
Photobiol 1998, 150–156.
20 Land EJ & Swallow AJ (1969) One-electron reactions in
biochemical systems as studied by pulse radiolysis. II.
Riboflavin. Biochemistry 8, 2117–2125.
21 Mu

¨
ller F, Bru
¨
stlein M, Hemmerich P, Massey V &
Walker WH (1972) Light-absorption studies on neutral
flavin radicals. Eur J Biochem 25, 573–580.
22 El Hanine-Lmoumene C, Lindqvist L & Favaudon V
(1997) One-electron photo-oxidation of reduced
Desulfovibrio vulgaris flavodoxin on laser excitation at
355 nm. Biochim Biophys Acta 1339, 97–100.
23 Watenpaugh KD, Sieker LC & Jensen LH (1973) The
binding of riboflavin-5¢-phosphate in a flavoprotein:
flavodoxin at 2.0-A
˚
resolution. Proc Natl Acad Sci USA
70, 3857–3860.
24 Mu
¨
ller F (1987) Flavin radicals: chemistry and biology.
Free Radic Biol Med 3, 215–230.
25 Draper RD & Ingraham LL (1968) A potentiometric
study of the flavin semiquinone equilibrium. Arch
Biochem Biophys 125, 802–808.
26 Ehrenberg A, Mu
¨
ller F & Hemmerich P (1967) Basicity,
visible spectra, and electron spin resonance of
flavosemiquinone anions. Eur J Biochem 2, 286–293.
27 Mu
¨

ller F, Hemmerich P, Ehrenberg A, Palmer G &
Massey V (1970) The chemical and electronic structure
of the neutral flavin radical as revealed by electron spin
resonance spectroscopy of chemically and isotopically
substituted derivatives. Eur J Biochem 14, 185–196.
28 Vaish SP & Tollin G (1971) Flash photolysis of flavins.
V. Oxidation and disproportionation of flavin radicals.
J Bioenerg 2, 61–72.
29 Hazzard JT, McDonough CA & Tollin G (1994) Intra-
molecular electron transfer in yeast flavocytochrome b
2
upon one-electron photooxidation of the fully reduced
enzyme: evidence for redox state control of heme–flavin
communication. Biochemistry 33, 13445–13454.
30 Rao KS & Lederer F (1998) About the pK
a
of the
active-site histidine in flavocytochrome b
2
(yeast
l-lactate dehydrogenase). Protein Sci 7, 1531–1537.
31 Ghrir R & Lederer F (1981) Study of a zone highly
sensitive to protease in flavocytochrome b
2
from
Saccharomyces cerevisiae. Eur J Biochem 120, 279–287.
32 Massey V (2002) The reactivity of oxygen with
flavoproteins. Intern Congress Series 1233, 3–11.
33 Massey V (1994) Activation of molecular oxygen by
flavins and flavoproteins. J Biol Chem 269, 22459–22462.

34 Fleischmann G, Lederer F, Mu
¨
ller F, Bacher A &
Ru
¨
terjans H (2000) Flavin–protein interactions in
flavocytochrome b
2
as studied by NMR after
reconstitution of the enzyme with
13
C- and
15
N-labelled
flavin. Eur J Biochem 267, 5156–5167.
35 Daff S, Ingledew WJ, Reid GA & Chapman SK (1996)
New insights into the catalytic cycle of flavocytochrome
b
2
. Biochemistry 35, 6345–6350.
L. Lindqvist et al. Flavin semiquinone protolysis
FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS 971
36 Pompon D (1980) Flavocytochrome b
2
from baker’s
yeast. Computer-simulation studies of a new kinetic
scheme for intramolecular electron transfer. Eur J
Biochem 106, 151–159.
37 Pompon D, Iwatsubo M & Lederer F (1980)
Flavocytochrome b

2
(baker’s yeast). Deuterium isotope
effect studied by rapid-kinetic methods as a probe for
the mechanism of electron transfer. Eur J Biochem 104,
479–488.
38 Chapman SK, Reid GA, Daff S, Sharp RE, White P,
Manson FDC & Lederer F (1994) Flavin to haem
electron transfer in flavocytochrome b
2
. Biochem Soc
Trans 22, 713–718.
39 Kellmann A, Lindqvist L, Tfibel F & Guglielmetti R
(1986) Nanosecond laser photolysis of the reaction
mechanism in the photochromism of piperidinospyropy-
ran. J Photochem 35, 155–167.
40 Amand B & Bensasson R (1975) Determination of
triplet quantum yields by laser flash absorption
spectroscopy. Chem Phys Lett 34, 44–48.
41 Rouvie
`
re N, Mayer M, Tegoni M, Capeille
`
re-Blandin C
& Lederer F (1997) Molecular interpretation of
inhibition by excess substrate in flavocytochrome b
2
:a
study with wild-type and Y143F mutant enzymes.
Biochemistry 36, 7126–7135.
42 Tegoni M, Janot JM & Labeyrie F (1990) Inhibition

of l lactate cytochrome c reductase – flavocytochrome
b
2
– by product binding to the semiquinone transient:
loss of reactivity towards monoelectronic acceptors.
Eur J Biochem 190, 329–342.
Flavin semiquinone protolysis L. Lindqvist et al.
972 FEBS Journal 277 (2010) 964–972 ª 2010 The Authors Journal compilation ª 2010 FEBS