Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.     

Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.     

Laser-flash-photolysis studies of p-cresol methylhydroxylase. Electron-transfer properties of the flavin and haem components.

p-Cresol methylhydroxylase, a heterodimer consisting of one flavoprotein subunit and one cytochrome c subunit, may be resolved into its subunits, and the holoenzyme may then be fully reconstituted from the pure subunits. In the present study we have characterized the reduction kinetics of the intact enzyme and its subunits, by using exogenous 5-deazariboflavin semiquinone radical generated in the presence of EDTA by the laser-flash-photolysis technique. Under anaerobic conditions the 5-deazariboflavin semiquinone radical reacts rapidly with the native enzyme with a rate constant approaching that of a diffusion-controlled reaction (k = 2.8 X 10(9) M-1 X s-1). Time-resolved difference spectra at pH 7.6 indicate that both flavin and haem are reduced initially by the deazariboflavin semiquinone radical, followed by an additional slower intramolecular electron transfer (k = 220 s-1) from the endogenous neutral flavin semiquinone radical to the oxidized haem moiety of the native enzyme. During the steady-state photochemical titration of the native enzyme at pH 7.6 with deazariboflavin semiquinone radical generated by light-irradiation the haem appeared to be reduced before the protein-bound flavin and was followed by the formation of the protein-bound anionic flavin radical. This result suggests that the redox potential of the haem is higher than that of the flavin, and that deprotonation of the flavin neutral radical occurred during the photochemical titration. Reduction kinetics of the flavoprotein and cytochrome subunits were also investigated by laser-flash photolysis. The protein-bound flavin of the isolated flavin subunit was reduced rapidly by the deazariboflavin semiquinone radical (k = 2.2 X 10(9) M-1 X s-1), as was the haem of the pure cytochrome c subunit (k = 3.7 X 10(9) M-1 X s-1). Flash-induced difference spectra obtained for the flavoprotein and cytochrome subunits at pH 7.6 were consistent with the formation of neutral flavin semiquinone radical and reduced haem, respectively. Investigation of the kinetic properties of the neutral flavin semiquinone radical of the flavoprotein subunit at pH 7.6 and at longer times (up to 5s) were consistent with a slow first-order deprotonation reaction (k = 1 s-1) of the neutral radical to its anionic form.     

Chicken liver sulfite oxidase. Kinetics of reduction by laser-photoreduced flavins and intramolecular electron transfer

Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin (dRFH.), lumiflavin (LFH.), and riboflavin (RFH.) semiquinone radicals with the redox centers of purified chicken liver sulfite oxidase. Kinetic studies of the native enzyme with dRFH. yielded a second-order rate constant of 4.0 X 10(8) M-1 s-1 for direct reduction of the heme and a first-order rate constant of 310 s-1 for intramolecular electron transfer from the Mo center to the heme. The reaction with LFH. gave a second-order rate constant of 2.9 X 10(7) M-1 s-1 for heme reduction. Reoxidation of the reduced heme due to intramolecular electron transfer to the Mo center gave a first-order rate constant of 155 s-1. The direction of intramolecular electron transfer using dRFH. and LFH. was independent of the buffer used for the experiment. The different first-order rate constants observed for intramolecular electron transfer using dRFH. and LFH. are proposed to result from chemical differences at the Mo site. Flash photolysis studies with cyanide-inactivated sulfite oxidase using dRFH. and LFH. resulted in second-order reduction of the heme center with rate constants identical with those obtained with the native enzyme, whereas the first-order intramolecular electron-transfer processes seen with the native enzyme were absent. The isolated heme peptide of sulfite oxidase gave only second-order kinetics upon laser photolysis and confirmed that the first-order processes observed with the native enzyme involve the Mo site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromatium flavocytochrome c: kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex

The kinetics of reduction of Chromatium vinosum flavocytochrome c heme subunit by exogenous flavin neutral semiquinones generated by laser flash photolysis have been investigated. Unlike the holoprotein, the isolated heme subunit was appreciably reactive with lumiflavin neutral semiquinone. The measured rate constant for the reaction (2.7 X 10(7) M-1 S-1) was comparable to those of c-type cytochromes having similar redox potentials. The ionic strength dependence of the reaction with FMN neutral radical indicated that the heme subunit had a small negative charge at the site of reduction. Taken together, these results suggest that the active site of the heme subunit is buried on complexation with the flavin subunit in the holoprotein. Horse cytochrome c formed a strong complex with Chromatium, but not Chlorobium, flavocytochrome c. Possible physiological electron acceptors such as HiPIP, cytochrome c', and cytochrome c-555 apparently did not bind to the flavocytochromes c. The rate constant for reduction by lumiflavin radical of horse cytochrome c complexed to flavocytochrome c was about twofold smaller than for reduction of horse cytochrome c alone. Flavocytochrome c was itself unreactive with exogenous flavin semiquinones. The ionic strength dependence of the reduction of the complex by FMN radical was also smaller than for horse cytochrome c in the absence of flavocytochrome c. Sulfite, which forms an adduct with the protein-bound FAD (FAD is bound in an 8-alpha-S-cysteinyl linkage), did not affect the reduction of horse cytochrome c in its complex with flavocytochrome c. We conclude that horse cytochrome c is reduced directly by exogenous flavins in its complex with flavocytochrome c, although the kinetics are slightly modified. These results are not unlike observations made with complexes of mitochondrial cytochrome c with cytochrome oxidase or cytochrome b5.     

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.     

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding.

Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron-transfer reactions of cytochrome f with flavin semiquinones and with plastocyanin. Importance of protein-protein electrostatic interactions and of donor-acceptor coupling.

Reduction of turnip ferricytochrome f by flavin semiquinones and oxidation of this ferrocytochrome f by French bean cupriplastocyanin are studied by laser flash photolysis over a wide range of ionic strengths. Second-order rate constants (+/- 15%) at extreme values of ionic strength, all at pH 7.0 and 22 degrees C, are as follows: with FMN semiquinone at 1.00 and 0.0040 M, 5.0 x 10(7) and 3.9 x 10(8) M-1 s-1; with riboflavin semiquinone at 1.00 and 0.0040 m, 1.7 x 10(8) and 1.9 x 10(8) M-1 s-1; with lumiflavin semiquinone at 1.00 and 0.0045 M, 1.8 x 10(8) and 4.5 x 10(8) M-1 s-1; with cupriplastocyanin at 1.00 and 0.100 M, 1.4 x 10(6) and 2.0 x 10(8) M-1 s-1. These reactions of cytochrome f are governed by the local positive charge of the interaction domain (the exposed heme edge), not by the overall negative charge of the protein. Lumiflavin semiquinone behaves as if it carried a small negative charge, probably because partial localization of the odd electron gives this electroneutral molecule some polarity; local charge seems to be more important than overall charge even for relatively small redox agents. The dependence of the rate constants on ionic strength was fitted to the equation of Watkins; this model recognizes the importance of local charges of the domains through which redox partners interact. There is kinetic evidence that a noncovalent complex between cytochrome f and plastocyanin exists at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd 1 nitrite reductase: regulation of the NO-bound end product

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.     

Direct measurement of intramolecular electron transfer between type I and type III copper centers in the multi-copper enzyme ascorbate oxidase and its type II copper-depleted and cyanide-inhibited forms

Transient kinetics of reduction of zucchini squash ascorbate oxidase (AO) by lumiflavin semiquinone have been studied by using laser flash photolysis. Second-order kinetics were obtained for reduction of the type I copper with a rate constant of 2.7 X 10(7) M-1 s-1, which is comparable to that obtained with other blue copper proteins such as plastocyanin. Following reduction, the type I copper was reoxidized in a protein concentration independent (i.e., intramolecular) reaction (kobs = 160 s-1). Comparison with literature values for limiting rate constants in transient single-turnover kinetic experiments suggests that intramolecular electron transfer probably is the rate-limiting step in enzyme catalysis. The extent of reoxidation of type I copper was approximately 55%, which is consistent with the approximately equal redox potentials of the type I and type III copper centers. Neither azide nor fluoride caused any significant changes in kinetics, although they are enzyme inhibitors and are thought to bind to the type II copper. In contrast, cyanide caused a concentration-dependent decrease in the extent of intramolecular electron transfer (with no change in rate constant), and decreased the rate constant for reduction of the type I copper by a factor of 2. The apparent dissociation constant for cyanide (0.2-0.4 mM) is similar to that reported for inhibition of enzyme activity. Removal of the type II copper from AO only marginally affected the kinetics of electron transfer to type I copper (k = 3.2 x 10(7) M-1 s-1) and slightly increased the extent but did not alter the rate constant of intramolecular electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro kinetics of reduction of cytochrome c554 isolated from the reaction center of the green phototrophic bacterium, Chloroflexus aurantiacus

The photochemical reaction center in the green bacterium Chloroflexus aurantiacus is similar to that found in purple phototrophic bacteria and interacts with a multiheme membrane-bound cytochrome. We have examined the kinetics of reduction of the pure solubilized reaction center cytochrome by laser flash photolysis of solutions containing lumiflavin or FMN. Reduction by lumiflavin semiquinone followed single exponential kinetics and the observed rate constant (kobs) was linearly dependent on protein concentration (k = 1.8 X 10(7) M-1s-1 heme-1). This result suggests either that the four hemes have similar reduction rate constants which cannot be resolved or that there are large differences in rate constant and only the most reactive heme (or hemes) was observed under these conditions. To determine the relative reactivities of the four hemes, we varied the extent of heme reduction at a single total protein concentration. As the hemes were progressively reduced by steady-state illumination prior to laser flash photolysis, kobs for the reaction with fully reduced lumiflavin decreased nonlinearly. Second-order rate constants for the four hemes were assigned by nonlinear least-squares analysis of kobs vs oxidized heme concentration data. The second-order rate constants obtained in this way for the highest and lowest potential hemes differed by a factor of about 20, which is larger than expected for c-type cytochromes based on redox potential alone (a factor of about 3 would be expected). This is interpreted as being due to differences in steric accessibility. Relative to the highest potential heme, which is as reactive as a typical c-type cytochrome, we estimated a steric effect of approximately twofold for heme 2, and steric effects of approximately fivefold for hemes 3 and 4. Using fully reduced FMN as reductant of oxidized cytochrome, ionic strength effects indicate a minus-minus interaction, with approximately a -2 charge near the site of reduction of the highest potential heme.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flavocytochromes c: transient kinetics of photoreduction by flavin analogues

Kinetics of reduction of phototrophic bacterial flavocytochromes c by exogenous flavin semiquinones and fully reduced flavins generated by laser flash photolysis have been studied. The mechanisms of reduction of Chromatium and Chlorobium flavocytochromes c are more similar to one another than previously thought. Neither protein is very reactive with neutral flavin semiquinones (k less than 10(7) M-1 s-1), and the reactions with fully reduced flavins are slower than expected on the basis of comparison with other electron-transfer proteins of similar redox potentials. Deazaflavin radical is reactive with the flavocytochromes c by virtue of its low redox potential, but this reaction is also slower than expected on the basis of comparison with other electron-transfer proteins. These experiments indicate that the active site for reduction of flavocytochrome c is relatively buried and probably inaccessible to solvent. Fully reduced FMN does not show an ionic strength effect in its reaction with flavocytochrome c, which demonstrates that the active site is uncharged. Sulfite, which forms an adduct with protein-bound FAD, partially blocks heme reduction. This shows that heme is reduced via the FAD. The rate constant for intramolecular electron transfer between FAD and heme must be on the order of 10(4) s-1 or larger.     

Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.     

Laser flash photolysis study of intermolecular and intramolecular electron transfer in trimethylamine dehydrogenase

Laser flash photolysis has been used to investigate the kinetics of reduction of trimethylamine dehydrogenase by substoichiometric amounts of 5-deazariboflavin semiquinone, and the subsequent intramolecular electron transfer from the FMN cofactor to the Fe4S4 center. The initial reduction event followed second-order kinetics (k = 1.0 x 10(8) M-1 s-1 at pH 7.0 and 6.4 x 10(7) M-1 s-1 at pH 8.5) and resulted in the formation of the neutral FMN semiquinone and the reduced iron-sulfur cluster (in a ratio of approximately 1:3). Following this, a slower, protein concentration independent (and thus intramolecular) electron transfer was observed corresponding to FMN semiquinone oxidation and iron-sulfur cluster reduction (k = 62 s-1 at pH 7.0 and 30 s-1 at pH 8.5). The addition of the inhibitor tetramethylammonium chloride to the reaction mixture had no effect on these kinetic properties, suggesting that this compound exerts its effect on the reduced form of the enzyme. Treatment of the enzyme with phenylhydrazine, which introduces a phenyl group at the 4a-position of the FMN cofactor, decreased both the rate constant for reduction of the protein and the extent of FMN semiquinone production, while increasing the amount of iron-sulfur center reduction, consistent with the results obtained with the native enzyme. Experiments in which the kinetics of reduction of the enzyme were determined during various stages of partial reduction were also consistent with these results, and further indicated that the FMN semiquinone form of the enzyme is more reactive toward the deazariboflavin reductant than is the oxidized FMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transfer process in cytochrome oxidase after pulse radiolysis

The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis. With the decay of the MNA radical, the absorption at 445 and 605 nm, a characteristic to ferrous heme a of the oxidase, increased. The kinetic difference spectrum obtained was similar to that of the fully reduced minus the fully oxidized form of the oxidase, and was not different from that obtained in the reaction of the MNA radical with the mixed valence CO complex of the oxidase, where heme a3 is the CO-bound reduced form with heme a oxidized. This suggests that the absorption changes at 445 and 605 nm arise from the reduction of heme a, not heme a3. In order to elucidate the contribution of "visible" copper in this reaction, the absorption of the oxidase in the near-infrared region was measured. A decrease of the 830 nm band due to the reduction of visible copper was detected with a half-life of 5 microseconds. This absorption change obeyed pseudo-first order kinetics and its rate constant increased with the concentration of the oxidase. This suggests that the absorption change at 830 nm is followed by a bimolecular reaction of the MNA radical with visible copper of the oxidase. After the first phase of the reduction, the return of the 830 nm band corresponding to oxidation of the copper was observed with a half-life of 100 microseconds. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme a increased. The rates of oxidation of the copper were identical to those of the reduction of heme a and independent of the oxidase concentration. This suggests that the MNA radical reacts with visible copper of the oxidase with a second order rate constant of 1.5 X 10(9) m-1 s-1 and subsequently the electron flows to heme a by intramolecular electron migration with a first order rate constant of 1.8 X 10(4) s-1. An activation energy of the intramolecular electron transfer was calculated to be 2.8 kcal/mol in the range 4-33 degrees C.     

Transient kinetics of flavin-photosensitized oxidation of reduced redox proteins. Comparison of c-type cytochromes and plastocyanins.

We have used laser flash photolysis to investigate the kinetics of oxidation of reduced plastocyanins obtained from spinach and the green alga Monoraphidium braunii by the triplet states of lumiflavin, riboflavin and FMN. We have compared the results of these experiments with the kinetics of reduction of the oxidized forms of these proteins by the corresponding flavin semiquinones, as well as with the kinetics of flavin oxidation and reduction of cytochrome c552 (Class I c-type cytochrome, generic name c553) from Monoraphidium. In all cases, the rate constants for oxidation were one or two orders of magnitude larger than for reduction, consistent with the greater thermodynamic driving force for the oxidation reaction. Similar steric and electrostatic effects were observed for both reactions with all proteins, suggesting that the same (or closely adjacent) sites were being utilized for electron removal and entry. The two algal proteins were quite similar to one another in their redox properties, consistent with their physiological role of being able to substitute for one another in photosynthetic electron transport. In contrast, the algal plastocyanin was more reactive than the spinach protein in both oxidation and reduction, suggesting differences in their steric properties at the site of electron transfer.     

Laser flash photolysis studies of the reduction kinetics of NADPH:cytochrome P-450 reductase

The reduction kinetics of NADPH:cytochrome P-450 reductase have been investigated by the laser flash photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Transients observed at 470 nm at neutral pH indicated that the oxidized reductase was reduced via second-order kinetics with a rate constant of 6.8 X 10(7) M-1 s-1. The second-order rate constant corresponding to the formation of the protein-bound semiquinone (measured at 585 nm) was essentially the same as that obtained at 470 nm (7.1 X 10(7) M-1 s-1). Subsequent to this rapid formation of protein-bound semiquinone, a partial exponential decay was observed at 585 nm. The rate of this decay remained invariant with protein concentration between pH 5.0 and 7.0, and a first-order rate constant of 70 s-1 was obtained for this process. This is assigned to intramolecular electron transfer from FADH. to FMN. Prior reduction of the enzyme to the one-electron level led to a decrease in both the second-order rate constant for reduction (2 X 10(7) M-1 s-1) and the first-order intraflavin electron transfer rate constant (15 s-1). The protein-bound FAD moiety of FMN-depleted reductase was reduced by 5-dRfH. with a second-order rate constant that was identical with that observed with the native enzyme (6.9 X 10(7) M-1 s-1). However, with this species no significant decay of the FAD semiquinone was observed at 585 nm following its rapid formation, consistent with the above assignment of this kinetic process.(ABSTRACT TRUNCATED AT 250 WORDS)