Single electron reduction of cytochrome c oxidase compound F: resolution of partial steps by transient spectroscopy

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.     

Dynamics of protein-protein docking: cytochrome c and cytochrome c peroxidase revisited

The dynamics of the docking step in the electron transfer reaction between yeast cytochrome c peroxidase and iso-1-cytochrome c has been studied using the Brownian dynamics method. In particular we have calculated the bimolecular rate constant at which a specific complex, the xray crystalline complex, can form in solution by translational and rotational diffusion in a field of force. Complexation criteria have been assessed based on the simultaneous alignment of three atom-atom contacts, as well as alternative criteria. The proteins are able to align one or two contacts at remarkably high rates, in fact, at rates approaching the diffusion-controlled limit for two spheres reactive over their entire surfaces. Three contacts may align, and hence the specific complex may dock, at rates on the order of 10(8) M(-1) s(-1), which is quite representative of the experimental association rate constant for ET-competent complex(es). The formation of the specific complex is strongly influenced by the favorable electrostatic interaction between these proteins. It is striking that a specific protein-protein complex can form within one order of magnitude as fast as two spherical proteins can touch at any orientation. It remains plausible that the high ET tunneling rate in this system can take place through a single highly favorable specific complex using a single high efficiency pathway. Still the contribution from a nonspecific set of complexes is not ruled out, particularly considering the marginal reproduction of the ionic strength dependence in the formation of the xray complex.     

Tissue-specific regulation of cytochrome c oxidase efficiency by nucleotides

Cytochrome c oxidase from bovine heart and liver was reconstituted in liposomes in the absence or presence of nucleotides. Intraliposomal ADP, and to a smaller extent intraliposomal ATP, increased the respiratory activity of the heart but not of the liver isozyme under uncoupled but not under coupled conditions, leading to increased respiratory control ratios. In a preceding publication [Anthony, G., Reimann, A., & Kadenbach, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 1652-1656], the stimulatory effect of intraliposomal ADP could be related to interaction with the matrix domain of subunit VIa-h (heart type). The data suggest a regulatory effect of matrix nucleotides in heart and skeletal muscle mitochondria on the efficiency of energy transduction in COX.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase

Determining the way in which deleterious mutations interact in their effects on fitness is crucial to numerous areas in population genetics and evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last (synergistic epistasis), then the evolution of sex and recombination may be favored to facilitate the elimination of deleterious mutations. However, there is a severe shortage of relevant data. Three relatively simple experimental methods to test for epistasis between deleterious mutations in haploid species have recently been proposed. These methods involve crossing individuals and examining the mean and/or skew in log fitness of the offspring and parents. The main aim of this paper is to formalize these methods, and determine the most effective way in which tests for epistasis could be carried out. We show that only one of these methods is likely to give useful results: crossing individuals that have very different numbers of deleterious mutations, and comparing the mean log fitness of the parents with that of their offspring. We also reconsider experimental data collected on Chlamydomonas moewussi using two of the three methods. Finally, we suggest how the test could be applied to diploid species.     

The interfacial behavior of cytochrome c studied by pendant-drop technique

Malnutrition is common and often undiagnosed in affected patients, especially those in the hospital, and is associated with impaired organ function, increased morbidity, and prolongation of hospital stay. It should be recognized and treated appropriately, because artificial nutritional support in malnourished patients leads to improvement in nutritional status and clinical outcome. There are multiple methods to provide nutrition, some by simply keeping the esophageal lumen patent, others by providing additional or all nutrients, including enteral and parenteral routes. The enteral route is preferred due to patient acceptance, lesser expense, and lower risk of complications. The addition of specific nutrients over standard diets may add benefit. Preoperative nutrition may reduce the risk of postoperative complications. Lastly, in the terminally ill patient, minimal intervention may be all that is needed to achieve the patient's comfort, perhaps the most important goal.     

Molecular dynamics simulation of cytochrome c3: studying the reduction processes using free energy calculations

The tetraheme cytochrome c3 from Desulfovibrio vulgaris Hildenborough is studied using molecular dynamics simulation studies in explicit solvent. The high heme content of the protein, which has its core almost entirely made up of c-type heme, presents specific problems in the simulation. Instability in the structure is observed in long simulations above 1 ns, something that does not occur in a monoheme cytochrome, suggesting problems in heme parametrization. Given these stability problems, a partially restrained model, which avoids destruction of the structure, was created with the objective of performing free energy calculations of heme reduction, studies that require long simulations. With this model, the free energy of reduction of each individual heme was calculated. A correction in the long-range electrostatic interactions of charge groups belonging to the redox centers had to be made in order to make the system physically meaningful. Correlation is obtained between the calculated free energies and the experimental data for three of four hemes. However, the relative scale of the calculated energies is different from the scale of the experimental free energies. Reasons for this are discussed. In addition to the free energy calculations, this model allows the study of conformational changes upon reduction. Even if the precise details of the structural changes that take place in this system upon individual heme reduction are probably out of the reach of this study, it appears that these structural changes are small, similarly to what is observed for other redox proteins. This does not mean that their effect is minor, and one example is the conformational change observed in propionate D from heme I when heme II becomes reduced. A motion of this kind could be the basis of the experimentally observed cooperativity effects between heme reduction, namely positive cooperativity.     

Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase

Elucidating the structures of intermediates in the reduction of O2 to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O2 with the mixed-valence enzyme, in which only heme a3 and CuB in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O==O bond cleavage occurs within the first 200 micros after reaction initiation; the presence of a uniquely stable Fe---O---O(H) peroxy species is not detected. The product of this rapid reaction is a heme a3 oxoferryl (FeIV==O) species, which requires that an electron donor in addition to heme a3 and CuB must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T., et al. Science, in press; Ostermeier, C., Harrenga, A. , Ermler, U. & Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547-10553] show that one of the CuB ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O---O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His---Tyr species to produce the product oxoferryl species, CuB2+---OH-, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O---O bond forming chemistry that occurs during O2 evolution in photosynthesis.     

Structural organization in peptide fragments of cytochrome c by heme binding

Prosthetic groups are often important structural organizers of proteins as well as essential functional components. Insertion of prosthetic groups is usually spontaneous, and implies an apoprotein that is partially preorganized to provide a recognition surface for specific binding. Cytochrome c is distinguished by having its heme attached by a dedicated heme lyase through thioether links to cysteine side-chains, and the apoprotein shows no evidence of preorganization under physiological conditions. Nevertheless, addition of heme to two short fragments of cytochrome c enhances helical structure substantially (from approximately 8% to approximately 22%), an effect that depends on iron ligation but not thioether linkage. The helical segments in the corresponding parts of the native holoprotein have little contact surface with heme, implying that the increased helical structure in the fragment complex may depend on tertiary interactions. The absence of the intervening polypeptide chain suggests that the complex represents a relatively independent folded subdomain.     

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.     

Different effects of carboxy-terminal deletion in the adrenodoxin molecule on cytochrome c and acetylated cytochrome c reductions

In immunoblotting analysis using a rabbit antibody to bovine adrenodoxin, the total proteins of the bovine adrenal cortex gave two bands, suggesting the presence of two forms of adrenodoxin in vivo: full-length and carboxy-terminal deleted adrenodoxins. To examine the effect of the carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrenodoxin were constructed. The wild type [Ad(2-128)] and carboxy-terminal deleted [Ad(2-114) and Ad(2-112)] recombinant adrenodoxins expressed in Escherichia coli were purified to give a single band on SDS-PAGE. They showed an A414/A276 value of 0.92. In an NADPH-cytochrome c reduction assay, the Km values for cytochrome c in the reconstituted system with AD(2-128), Ad(2-114) and Ad(2-112) were 39, 235 and 618 mM, respectively. The Vmax values were 638, 700 and 898 mol/min/mol flavin, respectively. In an NADPH-acetylated cytochrome c reduction assay, the maximum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maximum activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values were 4-times higher than that of Ad(2-128). In the presence of 100 mM NaCl, the Km values for acetylated cytochrome c in the system reconstituted with Ad(2-128), Ad(2-114) and Ad(2-112) were 220, 33 and 22 microM, respectively. The Vmax values were 352, 305 and 382 mol/min/mol flavin, respectively. These results indicate that the effects of the carboxy-terminal deletion of adrenodoxin on NADPH-cytochrome c and acetylated cytochrome c reductions are different; the carboxy-terminal region (residues 113-128) of adrenodoxin largely contributes to the binding with cytochrome c but disturbs the binding with acetylated cytochrome c.     

A stable, molten-globule-like cytochrome c

Expression of cytochrome c from Thermus thermophilus in Escherichia coli (E. coli) leads to a protein with characteristics of a molten globule. Unfolding induced by guanidine hydrochloride (GdHCl) shows that E. coli-expressed cytochrome c has lower stability (and less cooperativity of unfolding) compared to the protein extracted from Thermus thermophilus, even though the two proteins have identical amino-acid sequences. Moreover, Soret and far-UV circular dichroism signals differ for the two proteins, suggesting a distorted heme environment and more side-chain dynamics of E. coli-expressed cytochrome c. Still, tryptophan fluorescence in E. coli-expressed cytochrome c is quenched as in native protein, and the iron coordinates in a low-spin form. Amino-acid sequencing indicates the presence of only one covalent cysteine-linkage to the heme in E. coli-expressed cytochrome c (normally, there are two linkages), a possible explanation for the trapped, molten-globule-like structure. The features of this non-native protein may be of interest for interpretation of cytochrome c folding kinetics in vitro, since a molten globule may be an intermediate on the folding pathway.     

Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss

Activation of apoptosis is associated with generation of reactive oxygen species. The present research shows that superoxide is produced by mitochondria isolated from apoptotic cells due to a switch from the normal 4-electron reduction of O2 to a 1-electron reduction when cytochrome c is released from mitochondria. Bcl-2, a protein that protects against apoptosis and blocks cytochrome c release, prevents superoxide production when it is overexpressed. The switch in electron transfer provides a mechanism for redox signaling that is concomitant with cytochrome c-dependent activation of caspases. The block of cytochrome c release provides a mechanism for the apparent antioxidant function of Bcl-2.     

Multiple conformations of physiological membrane-bound cytochrome c

One-tenth of cytochrome c (cyt c) remains bound to the inner mitochondrial membrane (IMM) at physiological ionic strength (I; i.e. , I approximately 150 mM), exhibiting decreased electron transport (ET) activity. We now show that this form of membrane-bound cyt c (MB-cyt c) can be obtained in vitro and that binding to membranes at low I generates an additional conformation with higher ET activity. This low I bound form of MB-cyt c (MBL-cyt c) exhibited intrinsic ET rates similar to those of electrostatically bound cyt c (EB-cyt c). The ET activity of IMM-bound MB-cyt c approached slowly that of MBL-cyt c or EB-cyt c, suggesting that MB-cyt c converts to MBL-cyt c while bound to IMM. When maintained at physiological I, both forms of MB-cyt c were released from the membrane, indicating that they convert to an EB-cyt c-like form. This process may be very dynamic in cellular mitochondria, as binding and release for both MB-cyt c forms increased considerably with temperature. I-Dependent binding of MB-cyt c does not require IMM, and it can be reproduced using large or small unilamellar vesicles (SUV). Using SUV-cyt c complexes, we characterized the secondary structure of MB-cyt c and MBL-cyt c by circular dichroism. Conformational analysis revealed that cyt c binding as MB-cyt c decreases its alpha-helical content (70-79%) and increases its beta-sheet up to 135%. The secondary structure of MBL-cyt c was similar to that of EB-cyt c and soluble cyt c, with a modest increase in beta-sheet. Taken together, our experiments suggest that physiological cyt c exists in soluble and membrane-bound conformations with similar ET activity, which may exchange very rapidly, and that soluble hydrophilic proteins can bind transiently to biomembranes.     

Probing heart cytochrome c oxidase structure and function by infrared spectroscopy

IR spectra directly probe specific vibrators in bovine heart cytochrome c oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to Fe2+ a3 as two conformers each in isolated immobile environments sensitive to Fea and/or CuA oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN- that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fea, CuA, and CuB with associated changes in protein conformation of likely significance in O2 reduction and H(+)-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of alpha-helix (approximately 60%) and subtle changes with redox level and exposure to anesthetics. N2O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.     

Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.     

The mechanism of proton pumping by cytochrome c oxidasex127e comments

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the "peroxy" forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a3 propionates and are driven to the outside ("pumped") by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P --> F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F' with an alternative route to O.     

Characterization of cytochrome c free radical reactions with peptides by mass spectrometry

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.