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Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions

Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions
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  Fur. J. Biochem. 210, 577-583 (1992) CJ FEBS 1992 Effects of chemical modification of Anabaena flavodoxin and ferredoxin NADP reductase on the kinetics of interprotein electron transfer reactions Milagros MEDTNA , Carlos GOMEZ-MORENO and Gordon TOLLIN2 Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Spain Department of Biochemistry, University of Arizona, Tucson, USA (Received August 25, 1992) EJB 921223 The influcnce of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between PNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC 71 19. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred reduced Fd) by FNR,, (oxidized FNR) were dramatically decreased z 00-fold) when phenylglyoxal-modi- fied FNR was used, In contrast to the reaction involving the native proteins, no ionic strength effects on kohs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNR,, (FNR semiquinone) was used to reduce Fld,, (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fld,, (Fld semiquinone) formation via intracomplex electron transfer from FNR,, was approximately 12-fold smaller than that obtained for the native FNR (600 s vs 7000 s ). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate con- stant for intracomplex electron transfer from FNR,, to Fld,, which was approximately 7-fold smaller (1000 s- ) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved. Ferredoxin NADP reductase (FNR) is a flavoenzyme that plays a key role in the metabolism of photosynthetic organisms by catalyzing the photoreduction of NADP using ferredoxin (Fd) or flavodoxin (Fld) as electron donors. It has also been implicated in the reverse electron transfer from NADPH to nitrogenase via Fd or Fld that occurs in nitrogen- Correspondence t G. Tollin, Department of Biochcmistry, Uni- Fax: 1 602 621 9288. Abbreviations FNR, FNR,, and FNR,,, ferredoxin NADP reductase and its oxidized and one-electron-reduced seiniquinonc forms: Fld: Fld,, and Fld,,, flavodoxin and its oxidized and one- electron-reduced semiquinone forms; Fd and Fd, ferredoxin and its one-electron-reduced form; dRf and dRM , 5-deazariboflavin and 5-deazariboflavin semiquinone kobsr bserved pseudo-first-order rate constant; PG; phenylglyoxal; EtGly, glycine ethyl ester. versity of Arizona, Tucson, Arizona 85721, USA Enzyme Ferredoxin-NADP+ reductase (EC . fixing cyanobacteria, and its involvement in the light-depen- dent modulation of linear and cyclic electron transport, not only in cyanobacteria but also in higher plants, has been reported [l 21. The protein isolated from the cyanobacterium Anahaena PCC 7119 has a molecular mass of 36 kDa and contains one mole non-covalently bound FAD cofactor/mol enzyme. It has recently been crystallized and structural work using X-ray diffraction methods indicate that the crystals dif- fract up to 0.19-nm resolution [3]. Its amino acid sequence has been published [4] Chemical modification studies have indicated the involvement of argmine residues at both the NADP+ and the Fd binding sites of several FNR species [5, 61. In Anabuenu FNR: one arginine has been reported to be involved in the interaction with NADP , while a second such residue is apparently required for the binding with Fd [7]. Under physiological conditions the FNR from Anabaena PCC 71 19 cells grown at high levels of iron receives electrons  578 from Fd. This latter protein belongs to an important class of iron-sulfur protcins that function as electron transfer agents in biological processes such as photosynthesis, respiration, nitrogen fixation, and sulfur and carbon metabolism [8]. The iron-sulfur cluster is located near the surface of the molecule, in a region that carries an overall negativc charge and is highly conserved in other [2Fe-2S] ferredoxins [9]. In nahaena PCC 71 19, Fld is synthesized as a consequence of a lack of iron in the culture medium; it has been proposed that this flavoprotein replaces Fd, which is not present in the cells because of the iron limitation, in the transfer of electrons from photosystem I to FNR [lo, 111. The nahaena Fld has a molecular mass of 20 kDa, contains 1 mol FMN/mol protein, and forms a 1 1 complex with FNR from the same organism for which a Kd value of 8.5 pM at pH 8.0 has been determined [II, 121. In Fld, acidic amino acids predominate over basic ones, and it is generally assumed that some of these interact with positively charged residues on FNR. Indeed, chemical modification studies on the Fld from nabaena PCC 7119 have shown that Asp123 and/or Asp126, and Asp144 and/or Asp146, which are located in (or close to) the region where 'long-chain' flavodoxins show an insertion as compared to the 'short-chain' species, could play an essential role in substrate recognition [13]. Inasmuch as the requirement of charged amino acid resi- dues present in both FNR and Fld for the formation of func- tional electrostatic complexes has been demonstrated using steady-state and equilibrium methods, it is of considerable interest to investigate the involvement of these residues in the mechanism of the transfer of electrons within protein protein complexes using transient kinetic techniques. Laser flash photolysis has previously been used to monitor the kin- etics of reduction of protein -protein complexes and intermo- lecular electron transfer between FNR and Fld [14] and be- tween Fd and FNR [15] from nabaena PCC 7119. This technique is appropriate for this purpose since it involves the photochemical generation n s tu of a strong reductant (5- deazariboflavin semiquinone), which allows the measurement of electron transfer reactions that are too fast to be determined by rapid-mixing methods [16- 181. In the present paper we have used this approach to investigate the kinetics of reduction and intermolecular electron transfer between FNR modified by phenylglyoxal (PC) and Fld or Fd, and also between FNR and Fld modified by glycine ethyl ester (EtGly). The targets of modification of PG in nabaena PCC 71 19 FNR have been shown to be three specific arginine residues [19]. Two of these are implicated in the binding of NADP' (Arg224 and Arg233), and the other (Arg77) is indicated as being involved in the binding to Fd. As noted above, incubation of Fld with EtGly in the presence of a carbodimide has been reported to modify four of the aspartic acid residues on the surface of the protein (Asp123, Asp126, Asp144 and Asp146) [13]. The results presented here indicate that chemical modification of these specific residues in FNR and Fld produce very dramatic changes in the transient kinetics of the electron transfer reac- tions between the proteins, suggesting the involvement of these amino acids in the mechanisms of complex formation and intracomplex redox processes. MATERIALS AND METHODS Fld, FNR and Fd were isolated from nabaena PCC 71 19 as described by Pueyo and Gomez-Moreno [20]. FNR modi- fied by PG in the presence of NADP , and Fld modified by EtGly, were obtained as described by Sancho et al. [7] and by Medina et al. [I 91, respectively. 5-Deazariboflavin (dRf) was synthesized using the procedure of Smit et al. [21]. Absorption coefficients of the chemically modified proteins were almost indistinguishable from those reported for the native proteins [20]. All flash photolysis experiments were performed in 4 mM sodium phosphate buffer containing 0.5 mM EDTA, pH 7.0 I 10 mM . Ionic strengths greater than 10 mM were achieved by the addition of NaCl to this buffer. Buffer solu- tions were made anaerobic by bubbling with argon for at least 1 h before the addition of aliquots of concentrated enzyme stock. When necessary, trace amounts of oxygen introduced into the sample by this addition were removed by passing argon over the surface of the solution. Visible spectra were recorded on an On-Line Instrument Systems modification of a Cary 15 spectrophotometer. Laser flash photolysis experiments were performed anaer- obically at room temperature in the presence of approximately 90 pM dRf when EtGly-modified Fld was assayed, and 100 pM dRf when PG-modified FNR was assayed. Pro- cedures were as described in Walker et al. [14,15]. Photoexcita- tion of dRf was accomplished with a Photochemical Research Associates model LNIOO nitrogen laser which pumped a model LN102 dye laser using a BBQ dyc solution (PRA 2A386; emission wavelength, 395 nm; pulse duration approxi- mately 1 ns). The optical system used to monitor the reaction has been previously described [22]. Laser flash photolysis generated 5-deazariboflavin semiquinone (dRfH'), via reduction of the triplet flavin by EDTA; this is an unstable, strongly reducing, free radical species which either disproportionated [23] or, in the presence of a redox protein, rapidly underwent electron transfer to generate the reduced protein [I 71. When more than one protein is present simultaneously, the species which undergoes re- duction depends on the relative rate constants and the relative concentrations of the two proteins, and in the case of pre- formed complexes on the relative accessibility of the redox centers within the complex [14, 151 (see below for further discussion). All kinetic experiments were performed under pseudo-first-order conditions, in which the concentration of the protein acceptor 2 .5 pM) was in excess over the amount of dRfH' produced per flash < 0.5 pM . The laser flash inten- sity was approximately 0.1 mJ. In experiments such as these the process of protein reduction is always in competition with dRfH. disproportionation. The contribution of the dispro- portionation reaction to the observed transient decay kinetics is determined by the magnitude of the second-order rate con- stant for the protein reduction and the relative concentrations of the reacting species (i.e. protein vs dRfH'). In the present experiments, this complication was insignificant at the protein concentrations used, and we were able to fit the observed transients well with a single exponential whose magnitude was protein-concentration-dependent. Unless quantitation was re- quired, the number of flashes which were signal averaged to obtain a kinetic trace varied. Kinetic transients were analyzed by fitting the data to an exponential curve, ignoring the initial absorbance change due to dRf reduction. some cases, the data were analyzed using the program SIFIT (Olis Co.), yield- ing comparable results. The estimated error in the rate con- stant determinations was o , based upon standard de- viations from replicate measurements. Nonlinear protein concentration dependencies of kobs ob- served pseudo-first-order rate constant) values were obtained in experiments involving FNR in the presence of Fd or Fld, implying mechanisms which involved intermediate complex  579 [FNRl PM Fig 1. Concentration dependencies or thc laser-flash-induced rcduction of nabaena PCC 7 9 native 0, ) nd PGrnodified (A, A) FNR. Transients obtained at 461 nm a, A) and 600 nm 0, n mM sodium phosphate pH 7.0 containing 0.5 mM EDTA (ionic strength 10 mM). formation [24]. The apparent complex dissociation constant and the limiting first-order rate constant for intracomplex electron transfer can be evaluated from these data by a nonlinear least-squares computer-fitting procedure as de- scribed previously [25, 261. This yielded results for the native proteins which were comparable to those reported in the pre- vious studies [14, 151. RESULTS AND DISCUSSION Reduction of PG-modified ferredoxin NADP+ reductase from nabaena PCC 7119 by dRW The reduction of PG-modified nahaena FNR by laser- generated dRfJ3 was monitored by the absorbance decrease at 461 nm, which is in the region of maximum absorption for the oxidized FAD moiety of FNR [14]. This wavelength monitors both the formation and decay of dRfH and the reduction of FAD, and thus is convenient for following the redox chemistry initiated by the laser flash. At 10 mM ionic strength, laser photolysis resulted in a rapid initial increase in absorbance followed by an exponential decay that eventually went below the preflash baseline (data not shown). As was previously described [14, 151, the initial absorbance increase is due to the production of dRfH , while the net bleaching of absorbance is a result of the one-electron reduction of the FAD cofactor of FNR by dRfH . The corresponding forma- tion of the FAD semiquinone was also observed as an increase in absorbance at 600 nm (not shown). Transients were well fitted by monoexponential curves, and the observed pseudo- first-order rate constants obtained from the absorbance changes at the two wavelengths were identical (Fig. 1). The kohs values were linearly dependent on the concentration of FNR (Fig. l , giving a second-order rate constant of 1.7k0.3 lo8 M- s- at an ionic strength of 10 mM. This value is 2.5-fold smaller than that reported previously for the reduction of the native enzyme (4.2_f0.2x1OS M- s- ; cf. Fig. 1 and [15]), which suggests that the FAD cofactor of FNR is either sterically hindered or has its reduction potential significantly lowered by the chemical modification. The fact that the FAD environment is modified by the reaction with PG has already been reported, based on the observation that the absorption spcctrum of the modified enzyme is perturbed in the visible region [7]. From the crystal structure determination of spinach FNK [27), we can infer that one of the arginine residues of nabaena FNK which has been found to incorporate PG (Arg77; cf. [19]) s located in the FAD-binding domain as part of a surface loop which carries a positive electrostatic potential, and which is close to the exposed dimethylbenzene portion of the flavin cofactor. The side chain of the corresponding residue in the spinach enzyme (Arg93) has been reported to stabilize, via hydrogen bonding, the diphosphate moiety of the FAD pros- thetic group [27]. Chemical modification of this residue by PG could thus be the cause of both the distortion of the absorption spectrum of the protein in the visible region, and the lower FAD semiquinone formation rate constant upon reduction by dRfH . From the data presented here, we cannot determine if these effects are due to the proximity of the aromatic ring to the FAD cofactor, or to a conformational change in the protein induced by its reaction with PG. Reduction of EtGly-modified flavodoxin from nabaene PCC 7119 by dRfH The reduction of EtGly-modified nabaena Fld by laser- generated dRfH was monitored by the absorbance changes at 465 nm and 580 nm (cf. [14]). At 10 mM ionic strength, no significant differences were found between the kinetics of dRfH reduction of native and of EtGly-modified flavodoxin (data not shown). The fact that none of the residues which have been reported to be modified by EtGly [13] make immedi- ate contact with the FMN group, although they are in its vicinity, is consistent with this result. Reoxidation of reduced nabaena PCC 7119 ferredoxin by PG-modified ferredoxin- NADP reductase The kinetics of flash-induced reduction of nahaena Fd by dRfH in the presence of nahaena FNR have been previously described (cf. [15]). Within a preformed 1 I complex, re- duction occurs primarily at the FNR component, presumably because this has the most accessible redox center. In order to obtain Fd reduction, we use substoichiometric amounts of FNR, and thus electrons from dRfH are deposited primarily into Fd because of its higher concentration, followed by elec- tron transfer from Fdred o FNR,,. This is clearly demon- strated by an absorbance decrease at 507 nm, indicative of Fd,,d formation, followed by a reappearance of absorbance, which corresponds to the reoxidation of Fd,,d by FNR,, (Fig. 2a). Inasmuch as this wavelength is an isosbestic point for FNR reduction, any direct reduction of FNR by dRfJ3 does not contnbutc to the kinetic transient. An increase in absorbance at 600 nm due to FNR,, formation via interpro- tein electron transfer can also be observed in such experiments (data not shown; cf. [15]). At this wavelength, if direct FNK reduction occurs, its kinetics will be quite different from those due to interprotein electron transfer and can thus be easily distinguished. No ionic strength effects on the complex forma- tion constant for the nabaena proteins have been observed using this technique, whereas the rate constant for in- tracomplex electron transfer has a biphasic dependence on ionic strength, suggesting that electrostatic interactions help to orient the proteins appropriately, but that structural rear- rangements within the transient complex must occur to facili- tate electron transfer [1 51 During titration experiments in which aliquots of PG- modified FNR,, were added to a solution containing an excess of Fd,,, the observed rate constants at 10 mM ionic strength for electron transfer from Fdred o FNR,, obtained from laser flash transients collected at 507 nm (monitoring Fd reoxi- dation) and 600 nm (monitoring FNR reduction) displayed a  58 1.3ms I 267ms Fig. 2. Kinetic traces obtained at SO7 nm during the reduction of mix- tures of Anabaena PC 7119 Fd 37 pM) and FNR 10 pM) at 310 mM ionic strength. Arrow indicates time of laser flash. All othcr conditions as in Fig. 1. a) Native FNR. (b) PG-modified FNR. linear dependence on FNR concentration (shown at 507 nm in Fig. 3B). The kobs values for this intermolecular reaction are approximately 100-fold smaller than for the reaction with native FNR (Fig. 3A; also compare Fig. 2a and 2b). As re- ported previously [I 51, a non-linear protein concentration de- pendence is clearly observed with native FNR in the presence of Fd (Fig. 3A), which is ascribed to intermediate complex formation between the two proteins during electron transfer. The simplest interpretation of the present results with the PG-modified FNR is that the second-order rate constant for complex formation between Fdred and FNR,, has been dra- matically diminished by the chemical modification (the rate constant value calculated from Fig. 38 is 7.4 x lo5 M- s-l, whereas a minimal value of x I O M- s at 1 mM ionic strength can be estimated for native FNR from the data of Fig. 3A). As a consequence, the complex formation process is rate-limiting for interprotein electron transfer over the entire concentration range used in these experiments. Although we are unable to reach any conclusions concerning the effect of PG modification on the intracomplex electron transfer rate constant, the results clearly indicate that the modification of at least one of the arginines on the FNR surface by PG has an important effect on the binding of Fd to FNR, presumably due to the involvement of this residue in the protein-protein interaction. This conclusion is consistent with absorption spectroscopy studies of the modification of nabaena FNR by PG in the presence of NADP , which have indicated that a single, slowly modified, arginine residue is involved in the ferredoxin binding process [7]. As noted above, three arginine residues in FNR are modified by PG (Arg 77, Arg224 and Arg233; cf. [19]). The structural work on spinach FNR [27] implicates Arg77 (Arg93 in spinach) as being in the ferredoxin binding region, whereas the other two arginine residues are probably involved in NADP+ binding. Arg77 and the region in which it is located are highly conserved in other reductases [38] and previous chemical modification studies [29 31 have placed thc ferredoxin binding domain of the FNK in the cleft between the two domains of the protein, where Arg77 is presumably located in nabaena FNR. It thus seems likely [FNRI bM Fig 3. Reduction of native and PG-modified Anabaena FNR,, by Fdred. A) Concentration dependence of kobs values obtaincd for the re- duction of native nabaena FNR,, by Fdred t a) 10 mM and 0) 310 mM ionic strengths. B) Concentration dependence oPkObs alues obtained for the reduction of PG-modified nabaena FNR,, by Fdred at 0) 0 mM and e) 310 mM ionic strengths. Observed rate con- stants were obtained at 507 nm; dOx oncentration was 37 pM. All other conditions as in Fig. 1. that it is this arginine residue whose modification has caused the kinetic alterations described here. Further studies on FNR molecules containing alterations specifically located at this site will be required to confirm this conclusion. As noted above, the direct reduction of PG-modified FNR,, was only 2.5-fold slower than that obtained for the native protein. Thus, PG modification clearly has a much more significant effect on the Fd interaction than on the reaction with dRfH . This argues for the structural specificity of the kinetic effect. As was also observed with native FNR [15] no direct reduction of the PG-modified FNR,, by dRfH was detected in the presence of Fd, indicating a large decrease in the accessibility of the FAD center of the FNR, presumably due to complex formation with Fd,,. This latter result is also consistent with spectral studies [19], which have shown that PG-modified FNR,, can still form complexes with Fd,,, albeit with a 20-fold higher dissociation constant than with the native protein 60 pM vs 3.1 FM; determined in 100 mM so- dium phosphate pH 8.0). The data obtained for the PG-modified FNR at 310 mM ionic strength are identical to those obtained at 10 mM ionic strength (Fig. 3B), whereas the present data (Fig. 3A) as well as previous results with native FNR [IS] show a clear effect of salt concentration on kobs he absence of an ionic strength effect in the modified protein suggests that the altered arginine residue is an important contributor to the electrostatic interac- tion between the two proteins. Reduction of the complex between Anabaena PCC 71 19 PG-modified ferredoxin NADP reductase and flavodoxin The rate of interprotein electron transfer was also studied in a complex in which nabaena FNR is electrostatically bound to Anahaena Fld instead of to Fd. Previous stcady-  581 0 2 0 -0.2 -1.2 IIIII 1 0.3 0.6 0.9 1.2 1 5 0.15 0.15 i 0 4 4.4 1 0 1.2 2.4 3.6 4 8 6.0 Time ms) Fig. 4. Kinetic analyses of transients obtained upon laser photolysis of 1 : mixtures of nabaena FNR and flavodoxin. A) Transient obtained at 461 nm with PG-modified FNR and native Fld at 10 mM ionic strength. Protein concentrations were 25 pM. Solid line is a single exponential fit to the data k = 5.3+0.2 x lo3 s-I); residuals are shown in the upper box. Total absorbance decrease is 4.8 x 10 3; four flashcs averaged. (B) Transient obtained at 600 nm from solution in A). Solid line is a two-exponential fit to the data ( q = 6.1~1.2~10~s-~,k~ 5.7f0.3x102s~ );residualsareshownin the upper box. Total absorbance increase is 5.3 x six flashes averaged. (C) Transicut obtained at 600 nm with native Anabaena FNR and EtGly-modified Fld at 10 mM ionic strength. Protein con- centrations were 25 pM. Solid line is a two-exponential fit to the data k, = 1.07+0.1x104s~ , k2 = 9.6+0.3~10~s-~ ; esiduals are shown in the upper box. Total absorbance increase is 5.7 x tcn flashes averaged. state and transient kinetic experiments have shown that one- electron reduction of this complex results in the initial forma- tion of FNR,,, followed by intracomplex electron transfer to generate Fld,, [14]. The kinetics of the reduction of the PG- modified FNR- Fld electrostatic complex formed at I0 mM ionic strength were monitored at both 461 nm and 600 nm. When the PG-modified FNR ld complex was reduced by dRfH a monoexponential decay of absorbance at 461 nm to below the pre-flash baseline was observed, similar to that obtained for the native complex at this wavelength (Fig. 4A; cf. also [14]). The concentration dependence of the observed rate constant was determined by the addition of increasing equimolar concentrations of Fld and PG-modified FNR (Fig. 5A). A second-order rate constant of 2.3 50.2 x 10 M- s-l was obtained from such data which is, within experimental error, the same as that for the reduction of free PG-modified FNR by dM (see above) and significantly larger than that obtained previously for Fld alone 4r1 {FNRFId] bM) Fig. 5. Reduction of Anabaena flavodoxin by native or PG-modified Anabaena FNR. A) Concentration dependence of kobs alues obtained at 10mM ionic strength for the reduction of the electrostatically stabilized 1:l complex between Anahaena Fld and FNR at 0) 461 nm and 0) 00 nm, and betwcen Anabaena Fld and PG-modified FNR at (A) 461 nm and (A) 00 nm. B) Concentration dependence of k,, values obtained at 600 nm and at 100 mM ionic strength for the reduction of the electrostatically stabilized 1.1 complex between Anahuena Fld and native FNR 0) nd between Anabaena Fld and PG-modified FNR 0). or the PG-modified FNR systems, the kobs values at 600 nm correspond to the slow kinetic phase. (1.6k0.1 x lo8 M-I s- ; [14] .Aswasthecasewiththenative complex [14], the agreement between this value and that for the reduction of free PG-modified FNR suggests that elec- trons are entering via the FNR component, and that little or no steric hindrance of the FAD of PG-modified FNR occurs in the presence of Fld. The kinetics of one-electron reduction of the PG-modified FNR - ld complex were also monitored at 600 nm (Fig. 4B). The laser flash was followed by a biphasic increase in absorbance due to the production of protein-bound flavin semiquinone (cf. [14]). The rapid phase of this transient gave rate constant values similar to those obtained at 461 nm. We thus attribute this phase to the formation of FNR,, upon reduction by dRfH . The observed rate constants for the slow phase of reduction obtained at this wavelength were essentially independent of protein concentration over the range used (Fig. 5A). Thus, this kinetic process can be associated with the intracomplex electron transfer from FNR,, to Fld,, (pre- sumably, the difference in the absorbancies of FNR,, and Fld,, at 600 nm is large enough that ths process can be ob- served; cf. [14]). According to this interpretation, the value of kobs btained for the slow phase with the PG-modified protein, and the limiting value obtained at high protein concentrations for the native protein, correspond to the first-order rate con- stant for this intracomplex electron transfer process. Note that this ratc constant for the PG-modified system is appreciably smaller (600 s-I than that obtained at 600 nm for the com- plex between the native proteins (7000 s- , as obtained from Fig. 5A and in [14]). We thus conclude that the intracomplex electron transfer reaction is much less efficient in the PG- modified system than in the native system. Consistent with this, the difference between the transients obtained at 461 nm and at 600 nm was more clearly evident in the experiments
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