Abbreviations FDH, flavodehydrogenase domain of flavocytochrome b 2 ; FMN•, anionic FMN semiquinone; FMNH, fully reduced FMN anion; FMNH•, neutral FMN semiquinone; LCHAO, recombinant lon
Trang 1L-a-hydroxyacid-oxidizing flavoenzymes – a study
using nanosecond laser flash photolysis
Lars Lindqvist1, Simona Apostol1,*, Chaibia El Hanine-Lmoumene1and Florence Lederer2
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 b2;
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: lwlindqvist@gmail.com
*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 b2 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 pKa 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 b2 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.
Trang 2another 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¨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 flavin radical without limitation of the observation
time The photochemical reaction was achieved by
two-photon excitation of the reduced flavin Indeed,
our previous studies [4,5] of FMNH)in aqueous
solu-tion showed that pulsed laser excitasolu-tion of the reduced
flavin 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 (eaq))
and the neutral FMN semiquinone (FMNH•):
FMNHþ 2hv ! FMNHþ eaq ð1Þ
In those experiments, the flavin radical appeared
initially as the neutral (blue) species (FMNH•) at pH
7-10; however, acid–base 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 flavoenzymes In this paper, we report results
obtained for two members of a flavoenzyme family that
oxidizes l-2-hydroxy acids: rat long-chain hydroxy acid
oxidase (LCHAO, EC 1.1.3.15, isozyme B) and the
flavodehydrogenase domain (FDH) of flavocytochrome
b2, 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 b8a8 fold
and around the flavin [8–10] Family members stabilize
the anionic semiquinone at physiological pH [11–15]
This has been demonstrated to be the case for
flavo-cytochrome b2and 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 pKa 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 flavoprotein solutions in the relevant buffer (see Fig 1 for details) flushed with argon were exposed to laser pulses of varying fluence The appearance of eaq ) was measured at the 715 nm absorption peak of this species [18] at the end of the laser pulse, and the eaq ) concentration was calculated using the extinction coef-ficient 1.85· 104m)1Æcm)1 [18] The results (Fig 1) revealed that eaq)is formed for both proteins, confirm-ing the occurrence of photoionization (Eqn 1) The
eaq) 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 flavin 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 fluence rates used here
As photoionization is a two-photon process, one would expect the eaq ) yield to increase quadratically with the laser fluence However, a previous study of free FMNH) in aqueous solution [5] showed that the formation of eaq) was proportional to the square of the laser fluence only at the lowest fluences, and then increased quasi-linearly with the fluence The deviation from a quadratic response was ascribed to depletion of ground-state flavin 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 eaq)was noticeable only above a certain fluence threshold and then increased almost linearly with the fluence
5 10
15
LCHAO
0 0.02 0.04 0.06 0
0 0.02 0.04 0
2 4 6
FDH
FMNH·
Fig 1 Yields of eaq)and FMNH • obtained upon laser excitation at the end of the laser pulse for the FDH domain (70 l M in 25 m M
Tris ⁄ H 2 SO4, pH 7.8) and LCHAO (150 l M in 10 m M Tris ⁄ HCl,
pH 7.5).
Trang 3The formation of FMNH• at the end of the laser
pulse was measured at 570 nm in N2O-saturated
solutions to scavenge eaq) FMNH• concentrations
were obtained using the extinction coefficient
5· 103 m)1Æcm)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
eaq), increases almost linearly with the laser fluence;
however, the FMNH•concentration is higher than that
of eaq), 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 eaq) yields gave a considerably
lower value, approximately 60%, for the protein
com-pared to that for free FMNH) The deficit in eaq)yield
for the two proteins may be explained by assuming
that part of the eaq) 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 eaq) 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 eaq)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 N2O-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· 103m)1Æcm)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
300 400 500 600 700
300 400 500 600 0
4000 8000 12000 16000
Wavelength (nm)
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 N2O-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.
Trang 4evolution 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 (DAend, after 1–5 ls), normalized with
respect to the absorbance variation at the end of the
laser pulse (DA0), 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 pKa 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 pKa= 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
pKa 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 (k1
and k2, respectively) reveal trends in the pH
depen-dence of the deprotonation rates (Table 1) For the FDH domain, k1 increases from pH 7.7 to 8.5 For LCHAO, the ‘fast’ phase is absent at pH 7.5, reflecting the higher pKa 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
0.0 0.2 0.4 0.6 0.8
1.0
LCHAO
pKa = 8.7
FDH
pKa = 8.1
Aend
A0
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 (DAend) to that at the moment of the laser pulse (DA0) is plotted against pH The smooth curves were obtained from the expression for DA end ⁄ DA 0 expected at protolytic equilibrium:
DAend⁄ 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)
0.00
0.01
0.02
0.03
FDH
pH 8.3
pH 7.7
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 k1(· 10 6 s)1) k2(· 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
Trang 5As 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 (k2) of the ‘slow’ phase is
approxi-mately the same for the two proteins (2–3· 104s)1) at
the lowest pH (pH 7.5-7.7), even when the ‘fast’ phase
is absent At higher pH, the k2 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, H2O 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 H2O as possible proton acceptors:
FMNHþ Tris ! FMNþ TrisHþ ð2Þ
FMNHþ H2O! FMNþ H3Oþ ð3Þ
The contribution of Tris buffer to the deprotonation
was determined by studying the effect of its
concentra-tion on the ‘fast’ deprotonaconcentra-tion 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 k1=
k0+ kTris· [Tris], where kTris is approximately 1.4· 106m)1Æs)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· 107m)1Æs)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 k0of 1.4· 105s)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
pKa 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 b2 by electron transfer from FMNH) to heme b2 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· 106
m)1Æs)1and 1.4· 105s)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 H2O as the proton acceptor and a pKaof)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
10 m M Tris
100 m M Tris
Time (µs)
0.1 0.2 0.3
k1
[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 N2O) buffered at pH 7.7 with 10 or 100 m M 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 k1= (1.4 · 10 5 + 1.4 · 10 6 · [Tris])
s)1.
Trang 6obtained 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 pKa of the tyrosines
is not known, but it has been determined for
flavocyto-chrome b2 that the active site histidine (H373) has an
abnormal pKa of 9.1 in the reduced enzyme [30] The
crystal structure suggests that the pKaof 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 pKa 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 pKa 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 pKa values associated with the
fast deprotonation phase would then correspond to the
pKa of the half-reduced flavin before conformational
relaxation A lower pKa 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 b2 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 b2 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 (25C), conditions close to those used here, the heme reduction rate was estimated to be of the order of 1.5· 103s)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
Trang 7Experimental 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
pulsed Xe UV lamp was used as the probing light source,
in crossed-beam configuration Samples under study were
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
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
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
The recombinant FDH domain was prepared as described
previously [12] For the laser flash experiments, 0.06-0.12 mm
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
the solution surface over 1 h on ice with gentle rocking The cuvettes were then closed by means of a septum, and
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 lL of 30 mm 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
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
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