Kinetics of intramolecular electron transfer in cytochrome bo3 from Escherichia coli

We have examined the temperature dependence of the intramolecular electron transfer (ET) between heme b and heme o(3) in CO-mixed valence cytochrome bo(3) (Cbo) from Escherichia coli. Upon photolysis of CO-mixed valence Cbo rapid ET occurs between heme o(3) and heme b with a rate constant of 2.2 x 10(5) s(-1) at room temperature. The corresponding rate of CO recombination is found to be 86 s(-1). From Eyring plots the activation energies for these two processes are found to be 3.4 kcal/mol and 6.7 kcal/mol for the ligand binding and ET reactions, respectively. Using variants of the Marcus equation the reorganization energy (lambda), electronic coupling factor (H(AB)), and the ET distance were found to be 1.4 +/- 0.2 eV, (2 +/- 1) x 10(-3) eV, and 9 +/- 1 A, respectively. These values are quite distinct from the analogous values previously obtained for bovine heart cytochrome c oxidase (CcO) (0.76 eV, 9.9 x 10(-5) eV, 13.2 A). The differences in mechanisms/pathways for heme b/heme o(3) and heme a/heme a(3) ET suggested by the Marcus parameters can be attributed to structural changes at the Cu(B) site upon change in oxidation state as well as differences in electronic coupling pathways between Heme b and heme o(3).     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Rates and Equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse heme a --> CuA process were found to be 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.     

Activation volumes for intramolecular electron transfer in Escherichia coli cytochrome bo(3)

In this report we describe the activation volumes associated with the heme-heme electron transfer (ET) and CO rebinding to the binuclear center subsequent to photolysis of the CO-mixed-valence derivative of Escherichia coli cytochrome bo(3) (Cbo). The activation volumes associated with the heme-heme ET (k=1.2 x 10(5) s(-1)), and CO rebinding (k=57 s(-1)) are found to be +27.4 ml/mol and -2.6 ml/mol, respectively. The activation volume associated with the rebinding of CO is consistent with previous Cu X-ray absorption studies of Cbo where a structural change was observed at the Cu(B) site (loss of a histidine ligand) due to a change in the redox state of the binuclear center. In addition, the volume of activation for the heme-heme ET was found to be quite distinct from the activation volumes obtained for heme-heme ET in bovine heart Cytochrome c oxidase. Differences in mechanisms/pathways for heme b/heme o(3) and heme a/heme a(3) ET are suggested based on the associated activation volumes and previously obtained Marcus parameters.     

Structure of cytochrome c6A, a novel dithio-cytochrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications for function

Cytochrome c6A is a unique dithio-cytochrome present in land plants and some green algae. Its sequence and occurrence in the thylakoid lumen suggest that it is derived from cytochrome c6, which functions in photosynthetic electron transfer between the cytochrome b6f complex and photosystem I. Its known properties, however, and a strong indication that the disulfide group is not purely structural, indicate that it has a different, unidentified function. To help in the elucidation of this function the crystal structure of cytochrome c6A from Arabidopsis thaliana has been determined in the two redox states of the heme group, at resolutions of 1.2 A (ferric) and 1.4 A (ferrous). These two structures were virtually identical, leading to the functionally important conclusion that the heme and disulfide groups do not communicate by conformational change. They also show, however, that electron transfer between the reduced disulfide and the heme is feasible. We therefore suggest that the role of cytochrome c6A is to use its disulfide group to oxidize dithiol/disulfide groups of other proteins of the thylakoid lumen, followed by internal electron transfer from the dithiol to the heme, and re-oxidation of the heme by another thylakoid oxidant. Consistent with this model, we found a rapid electron transfer between ferro-cytochrome c6A and plastocyanin, with a second-order rate constant, k2=1.2 x 10(7) M(-1) s(-1).     

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o3 and its transfer to CuB. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of -1.3+/-0.3 mL mol-1 and enthalpy change of 32+/-1.6 kcal mol-1. Subsequently, a volume increase of 2.9 mL mol-1 with an enthalpy change of -5.3+/-2.5 kcal mol-1 is observed with the lifetime of approximately 250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo3 oxidase indicating that the heme o3/CuB active site dynamics are affected by the redox state of heme b.     

Cytochrome bo(3) from Escherichia coli: the binding and turnover of nitric oxide

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo(3)(cyt bo(3)) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo(3) was determined using an NO electrode in the [NO] range 1-14 microM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo(3) corresponded to binding of 1 NO molecule per cyt bo(3) functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption ( approximately 0.3molNOmol bo(3)(-1)min(-1)), indicating that cyt bo(3) possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1-14 microM) both fast and pulsed oxidised cyt bo(3) bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K(d)=5.6+/-0.6 microM and that NO binding was inhibited by the presence of Cl(-). The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo(3). These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to Cu(B)(II) results in the formation of the nitrosonium (Cu(B)(I)-NO(+)) complex. NO(+) then reacts with OH(-), a Cu(B) ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo(3) does result in the reduction of Cu(B).     

Nitric oxide reacts with the ferryl-oxo catalytic intermediate of the CuB-lacking cytochrome bd terminal oxidase

Cytochrome bd is a bacterial respiratory oxidase carrying three hemes but no copper. We show that nitric oxide (NO) reacts with the intermediate F of cytochrome bd from Azotobacter vinelandii: (i) with a 1:1 stoichiometry, (ii) rapidly (k=1.2 +/- 0.1 x 10(5)M(-1)s(-1) at 20 degrees C), and (iii) yielding the oxidized enzyme with nitrite bound to heme d at the active site. Unexpectedly, the NO reaction mechanism of this catalytic intermediate in the Cu(B)-lacking cytochrome bd appears similar to that of beef heart cytochrome c oxidase, where Cu(B) was proposed to play a key role.     

Site-directed mutagenesis of proline 94 to alanine in amicyanin converts a true electron transfer reaction into one that is kinetically coupled

Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro(94) resides in the "ligand loop" of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (H(AB)) and reorganization energy (lambda) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of H(AB) and lambda are 8.3 cm(-)(1) and 2.3 eV, respectively, compared to values of 0.3 cm(-)(1) and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry 43, 9372-9380]. In one of these, conformation B, a water molecule has replaced Met(98) as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 A. Analysis of these structures suggests that the true k(ET) for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu(2+) by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu(1+) by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the K(eq) and values of DeltaH degrees and DeltaS degrees that are associated with the non-ET reaction that modulates the observed ET rate.     

Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase

The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.     

Filling the catalytic site of cytochrome c oxidase with electrons. Reduced CuB facilitates internal electron transfer to heme a3

In the reductive phase of its catalytic cycle, cytochrome c oxidase receives electrons from external electron donors. Two electrons have to be transferred into the catalytic center, composed of heme a(3) and Cu(B), before reaction with oxygen takes place. In addition, this phase of catalysis appears to be involved in proton translocation. Here, we report for the first time the kinetics of electron transfer to both heme a(3) and Cu(B) during the transition from the oxidized to the fully reduced state. The state of reduction of both heme a(3) and Cu(B) was monitored by a combination of EPR spectroscopy, the rapid freeze procedure, and the stopped-flow method. The kinetics of cytochrome c oxidase reduction by hexaamineruthenium under anaerobic conditions revealed that the rate-limiting step is the initial electron transfer to the catalytic site that proceeds with apparently identical rates to both heme a(3) and Cu(B). After Cu(B) is reduced, electron transfer to oxidized heme a(3) is enhanced relative to the rate of entry of the first electron.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2)

Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

How does reorganization energy change upon protein unfolding? Monitoring the structural perturbations in the heme cavity of cytochrome c

In several classes of proteins the redox center provides an additional intrinsic biophysical probe that could be used to study the protein structure and function. In present report reorganization energy (lambda, as a parameter describing electron transfer properties) was used to study the protein structural changes around the heme prosthetic group in cytochrome c (cyt c). We attempted to monitor the value of this parameter upon the unfolding process of cyt c by urea, during which it was increased sigmoidally from about 0.52 to 0.82 eV for native and unfold protein, respectively. Results indicate that by structural changes in the heme site, lambda provides a complementary tool for following the unfolding process. Assuming a reversible two-state model for cyt c unfolding, Delta G(H2O), Cm and m values were determined to be 8.32+/-0.7 kcal mol(-1), 1.53+/-0.19 kcalmol(-1)M(-1) and 5.03 M, respectively.     

Kinetic resolution of a tryptophan-radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.