Chromatium vinosum cytochrome c-552. Reduction by photoreduced flavins and intramolecular electron transfer

Cytochrome c-552 from Chromatium vinosum is an unusual heme protein in that it contains two hemes and one flavin per molecule. To investigate whether intramolecular electron transfer occurs in this protein, we have studied its reduction by external photoreduced flavin by using pulsed-laser excitation. This approach allows us to measure reduction kinetics on the mirosecond time scale. Both fully reduced lumiflavin and lumiflavin semiquinone radical reduce cytochrome c-552 with second-order rate constants of approximately 1.4 x 10(6) M-1s-1 and 1.9 x 10(8) M-1 s-1, respectively. Kinetic and spectral data and the results of similar studies with riboflavin indicate that both the flavin and heme moieties of cytochrome c-552 are reduced simultaneously on a millisecond time scale, with the transient formation of a protein-bound flavin anion radical. This is suggested to be due to rapid intramolecular electron transfer. Further, steric restrictions play an important role in the reduction reaction. Studies were conducted on the redox processes following photolysis of CO-ferrocytochrome c-552 in which the flavin was partly oxidized to resolve the kinetics of electron transfer between the heme and flavin of cytochrome c-552. Based on these results, we conclude that intramolecular electron transfer from ferrous heme to oxidized flavin occurs with a first-order rate constant of greater than 1.4 x 10(6) s-1.     

Glutamate 107 in subunit I of cytochrome bd from Escherichia coli is part of a transmembrane intraprotein pathway conducting protons from the cytoplasm to the heme b595/heme d active site

Cytochrome bd is a terminal quinol:O 2 oxidoreductase of the respiratory chain of Escherichia coli. The enzyme generates protonmotive force without proton pumping and contains three hemes, b 558, b 595, and d. A highly conserved glutamic acid residue of transmembrane helix III in subunit I, E107, was suggested to be part of a transmembrane pathway delivering protons from the cytoplasm to the oxygen-reducing site. When E107 is replaced with leucine, the hemes are retained but the ubiquinol-1-oxidase activity is lost. We compared wild-type and E107L mutant enzymes during single turnover using absorption and electrometric techniques with a microsecond time resolution. Both wild-type and E107L mutant cytochromes bd in the fully reduced state bind O 2 rapidly, but the formation of the oxoferryl species in the mutant is dramatically retarded as compared to the wild type. Intraprotein electron redistribution induced by the photolysis of CO bound to ferrous heme d in the one-electron-reduced wild-type enzyme is coupled to the membrane potential generation, whereas the mutant cytochrome bd shows no such potential generation. The E107L mutation also causes decrease of midpoint redox potentials of hemes b 595 and d by 25-30 mV and heme b 558 by approximately 70 mV. There are two protonatable groups redox-linked to hemes b 595 and d in the active site, one of which has been recently identified as E445, whereas the second group remains unknown. Here we propose that E107 is either the second group or a key residue of a proposed proton delivery pathway leading from the cytoplasm toward this second group.     

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.     

Discovery of the true peroxy intermediate in the catalytic cycle of terminal oxidases by real-time measurement

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O(2) to the fully reduced enzyme is followed by the fast (5 micros) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b(558) remains reduced while high spin heme b(595) is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O(2) by two electrons is sufficient to produce (hydro)peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe (3+)(d)-O-O-(H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the P(M) intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b(558) in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b(595) are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 micros; it decays into oxoferryl species due to oxidation of low spin heme b(558) that is linked to significant charge translocation across the membrane.     

Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies.

Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers. To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).     

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)     

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.     

Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.     

Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: ferryl and oxy-ferrous species dominate

The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.     

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.     

Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen.

To probe the functional role of a bound ubiquinone-8 in cytochrome bo-type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol-1 and dioxygen. Stopped-flow studies showed that anaerobic reduction of the wild-type and the bound ubiquinone-free (DeltaUbiA) enzymes with ubiquinol-1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6-dibromo-4-cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol-1 by eliminating the fast phase. Flow-flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b-to-heme o electron transfer occurs with a rate constant of approximately 1x10(4) s(-1) in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low-spin heme b in subunit I.     

Strong excitonic interactions in the oxygen-reducing site of bd-type oxidase: the Fe-to-Fe distance between hemes d and b595 is 10 A

Absorption and circular dichroism (CD) spectra of cytochrome bd from Escherichia coli have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J. P., Huang, H. W., Moenne-Loccoz, P., Konstantinov, A. A., and Gennis, R. B. (2001) Biochemistry 40, 8548-8556]. The absorption bands of ferrous heme b595 are absent from the spectrum of the dithionite-reduced E445A form of cytochrome bd. The difference between the spectra of the dithionite-reduced WT and E445A enzymes indicates that in the mutant, heme b595 is present but is not reducible by dithionite. Cytochrome bd reveals intense CD signals dominated by heme d, with almost no contribution from heme b595 or heme b558. The CD spectrum of the reduced wild type enzyme in the Soret band indicates strong excitonic interactions between ferrous heme d and ferrous heme b595, and these interactions are not observed in dithionite-reduced E445A mutant, in which heme b595 remains in the ferric state. Modeling the excitonic interactions in both absorption and CD spectra has been carried out, yielding an estimate of the Fe-to-Fe distance between heme d and heme b595 of about 10 A. The physical proximity supports the hypothesis that heme d and heme b595 can form a di-heme oxygen reducing site, a unique structure for respiratory oxidases.     

Intramolecular electron transfer from c heme to d1 heme in bacterial cytochrome cd1 nitrite reductase occurs over the same distances at very different rates depending on the source of the enzyme.

Intramolecular electron transfer over 12 A from heme c to heme d(1) was investigated in cytochrome cd(1) nitrite reductase from Pseudomonas aeruginosa, following reduction of the c heme by pulse radiolysis. The rate constant for the transfer is relatively slow, k = 3 s(-1). The present observations contrast with a corresponding rate of electron transfer, 1.4 x 10(3) s(-1), measured for cytochrome cd(1) from Paracoccus pantotrophus, though the relative positions of the two heme groups are the same in both enzymes. The rate of intramolecular electron transfer within the enzyme from P. aeruginosa was accelerated 10(4)-fold (1.4 x 10(4) s(-1)) by the binding of cyanide to the d(1) heme. A coordination change at the d(1) heme upon its reduction is suggested to be a major factor in determining the slow rate of electron transfer in the P. aeruginosa enzyme in the absence of cyanide.     

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The rate of internal heme-heme electron transfer in cytochrome C oxidase

Cytochrome c oxidase catalyzes the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of dioxygen to water. The process involves a number of intramolecular electron-transfer reactions, one of which takes place between the two hemes of the enzyme, hemes a and a3, with a rate of approximately 3 x 10(5) s(-1) (tau approximately 3 micros). In a recent report [Verkhovsky et al. (2001) Biochim. Biophys. Acta 1506, 143-146], it was suggested that the 3 x 10(5) s(-1) electron transfer may be controlled by structural rearrangements and that there is an additional electron transfer that is several orders of magnitude faster. In the present study, we have reinvestigated the spectral changes occurring in the nanosecond and microsecond time frames after photolysis of CO from the fully reduced and mixed-valence enzymes. On the basis of the differences between them, we conclude that in the bovine enzyme the microscopic forward and reverse rate constants for the electron-transfer reactions from heme a to heme a3 are not faster than approximately 2 x 10(5) and approximately 1 x 10(5) s(-1), respectively.     

Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b₅₉₅, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b₅₉₅ and heme b₅₅₈. It is concluded that 1) O₂ binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O₂ binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b₅₉₅, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm]?→?E107?→?E99 (heme b₅₉₅)?→?E445 (heme d)?→?oxygenated heme d.     

Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.     

Proton and electron pathways in the bacterial nitric oxide reductase

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.     

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)     

Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity

The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihydromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O(2). In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and R391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N',N'-tetramethyl-p-phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center where O(2) is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b(558), heme b(595), and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg(391) is close to Met(393), one of the axial ligands to heme b(558), it is to be expected that the R391A mutation might destabilize the reduced form of heme b(558). The fact that the midpoint potentials of heme d and heme b(595) are also significantly lowered in the R391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.