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: 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 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 . Dynamics of flavin semiquinone protolysis in L -a- hydroxyacid-oxidizing flavoenzymes – a study using nanosecond laser flash photolysis Lars Lindqvist 1 ,. 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