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.     

Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent.     

Promoted electron transfer of mitoxantrone binding with DNA by cytochrome c

A promoted electron transfer of an antitumor drug, mitoxantrone (MTX), intercalating into DNA duplex was successfully obtained upon addition of cytochromes c (cyt. c) in NaAc-HAc buffer solution (pH 4.5). The experimental results suggested that co-existence of MTX and cyt. c in the DNA helix is an important factor for accelerated electron transfer of MTX, where the promoter, cyt. c, operated smoothly through the DNA bridge. The UV/Vis spectroscopic experiments further confirmed the interaction process. Furthermore, a possible mechanism of such reaction was also discussed in this paper.     

A cytosolic cytochrome b5-like protein in yeast cell accelerating the electron transfer from NADPH to cytochrome c catalyzed by Old Yellow Enzyme

A 410-nm absorbing species which enhanced the reduction rate of cytochrome c by Old Yellow Enzyme (OYE) with NADPH was found in Saccharomyces cerevisiae. It was solubilized together with OYE by the treatment of yeast cells with 10% ethyl acetate. The purified species showed visible absorption spectra in both oxidized and reduced forms, which were the same as those of the yeast microsomal cytochrome b5. At least 14 amino acid residues of the N-terminal region coincided with those of yeast microsomal b5, but the protein had a lower molecular weight determined to be 12,600 by SDS-PAGE and 9775 by mass spectrometry. The cytochrome b5-like protein enhanced the reduction rate of cytochrome c by OYE, and a plot of the reduction rates against its concentration showed a sigmoidal curve with an inflexion point at 6x10(-8) M of the protein.     

Cytochrome b(5) is a major reductant in vivo of human indoleamine 2,3-dioxygenase expressed in yeast

The evolutionary relationship of indoleamine 2,3-dioxygenase (IDO) to some gastropod myoglobins suggests that IDO may undergo autoxidation in vivo such that one or more currently unidentified electron donors are required to maintain IDO heme iron in the active, ferrous state. To evaluate this hypothesis we have used yeast knockout mutants in combination with a recently developed yeast growth assay for IDO activity in vivo to demonstrate a role for cytochrome b(5) and cytochrome b(5) reductase in maintaining IDO activity in vivo.     

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.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Oxidative modification of quercetin by hemeproteins

The ability of a number of hemeproteins to oxidize the flavonoid quercetin has been shown. It was found that quercetin undergoes chemical modification in the presence of cytochrome c, myoglobin, and hemoglobin but not cytochrome b(5). In the range of investigated proteins the most effective oxidant appears to be cytochrome c. Chromatographic analysis of the reaction mixture revealed a number of quercetin oxidation products. The main oxidation product was purified and characterized by means of LC-MS and NMR analyses. It has a dimeric structure similar to the product of quercetin oxidation by horseradish peroxidase and is formed during radical-driven reactions. Our results indicate that a number of hemeproteins can react and modify biologically active flavonoids. However, these reactions might also lead to the generation of active species with deleterious consequences for the cellular macromolecules.     

The operation of the alternative electron-leak pathways mediated by cytochrome c in mitochondria

Low (1 x 10(-9)M) concentrations of cytochrome c inhibit H2O2 production in cytochrome c-depleted mitochondria, purified succinate-cytochrome c reductase (SCR) and antimycin A inhibited cytochrome c-depleted HMP. At higher concentration (2 x 10(-6)M), cytochrome c eliminates pre-existed H2O2 if feeding electrons to it by succinate. Cytochrome c also decreases the OH* produced by succinate-cytochrome c reductase oxidizing succinate. We conclude that the alternative electron-leak pathway mediated by cytochrome c operates very well. In the presence of antimycin A, ferrocytochrome c can suppress the generation of H2O2 in SCR system, but ferricytochrome c cannot. Similar results are obtained on the elimination of pre-existed H2O2 by cytochrome c. For hydroxyl radical, antimycin A abolishes the suppression caused by both ferrocytochrome c and ferricytochrome c. These results indicate that the reductive state of cytochrome c caused by electron-flow is necessary and sufficient for the operation of cytochrome c-mediated electron-leakage pathway.     

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.     

The heme domain of cellobiose oxidoreductase: a one-electron reducing system

Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases.     

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.     

Electron transfer from cytochrome c to cupredoxins

Electron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (ΔG ⁰) and the reorganization energy. Here, we characterized ET from Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA) and its designed mutants to cupredoxins, Silene pratensis plastocyanin (PC) and Acidithiobacillus ferrooxidans rusticyanin (RC), through measurement of pseudo-first-order ET rate constants (k _obs). The influence of the ΔG ⁰ value for ET from PA to PC or RC on the k _obs value was examined using a series of designed PA proteins exhibiting a variety of E _m values, which afford the ΔG ⁰ variation range of 58–399 meV. The plots of the k _obs values obtained against the ΔG ⁰ values for both PA–PC and PA–RC redox pairs could be fitted well with a single Marcus equation. We have shown that the ET activity of cytochrome c can be controlled by tuning the E _m value of the protein through the substitution of amino acid residues located in hydrophobic-core regions relatively far from the redox center. These findings provide novel insights into the molecular design of cytochrome c, which could be utilized for controlling its ET activity by means of protein engineering. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electron tunneling through Pseudomonas aeruginosa azurins on SAM gold electrodes

Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH₃(CH₂)_nSH:HO(CH₂)_mSH (n = m = 4, 6, 8, 11; n = 13, 15 m = 11) self-assembled-monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6-11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn47 caused by deprotonation of nearby surface residues.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

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.     

Purification and characterisation of periplasmic C-type cytochromes from Desulfovibrio desulfuricans (NCIMB 8372)

Periplasmic extract from Desulfovibrio desulfuricans (NCIMB 8372) was found to contain two different c-type cytochromes. One is tetraheme cytochrome c₃ and the other is monoheme cytochrome c₅₅₃. Cytochrome c₃ could be purified by a procedure involving only one chromatographic step, whereas cytochrome c₅₅₃ required several such steps. Cytochrome c₃ was found to have a relative molecular mass of 14300 and an isoionic point higher than 9. Analysis of the redox potentials indicated one heme at -260 mV and three hemes around -330 mV. Cytochrome c₅₅₃ had a relative molecular mass of 7200, an isoionic point higher than 9 and a redox potential of 0 mV.     

Electrochemistry of cytochrome c immobilized on cardiolipin-modified electrodes: A probe for protein–lipid interactions

Electrochemistry of cytochrome c (cyt c) immobilized on a cardiolipin (CL)/phosphatidylcholine (PC) film supported on a glassy carbon electrode was investigated using variable-frequency AC voltammetry. At low ionic strength, we observed two redox-active subpopulations characterized by distinct values of potential (E_1/2) and electron transfer rate constant (k_ET). At high ionic strength, only one subpopulation was detected, consistent with the existence of very stable cyt c–CL adducts, most probably formed by hydrophobic interactions between the protein and the fatty acid (FA) chains carried by CL. This subpopulation exhibits a comparatively high k_ET value (> 300 s^− 1) apparently changing with the structure of the FA chains of CL, i.e. 18:2(n − 6) or 14:0. Our study suggests that electrochemistry can be a useful technique for probing protein–lipid interactions, and more particularly the role played by the specific structure of the FA chains of CL on cyt c binding.     

The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H

Elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. Here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the Thermus thermophilus ribonuclease H, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an α-helix and two β-strands (51 folded residues). Using multidimensional NMR, we solved the structure of this intermediate mimic to an atomic resolution (backbone rmsd, 0.51 Å). It has a native-like backbone topology and shows some local deviations from the native structure, revealing that the structure of the folded region of an early folding intermediate can be as well defined as the native structure. The topological parameters calculated from the structures of the intermediate mimic and the native state predict that the intermediate should fold on a millisecond time scale or less and form much faster than the native state. Other factors that may lead to the slow folding of the native state and the accumulation of the intermediate before the rate-limiting transition state are also discussed.