Investigation of protonatable residues in Rhodothermus marinus caa 3 haem-copper oxygen reductase: comparison with Paracoccus denitrificans aa 3 haem-copper oxygen reductase

The Rhodothermus marinus caa ₃ haem-copper oxygen reductase contains all the residues of the so-called D- and K-proton channels, with the notable exception of the helix VI glutamate residue (Glu278^I in Paracoccus denitrificans aa ₃), being nevertheless a true oxygen reductase reducing O₂ to water, and an efficient proton pump. Instead, in the same helix, but one turn below, it has a tyrosine residue (Tyr256^I, R. marinus caa ₃ numbering), whose hydroxyl group occupies the same spatial position as the carboxylate group of Glu278^I, as deduced by comparative modelling techniques. Therefore, we proposed previously that this tyrosine residue could play an important role in the proton pathway. In this article we further study this hypothesis, by investigating the equilibrium thermodynamics of protonation in R. marinus caa ₃, using theoretical methodologies based on the structural model previously obtained. Control calculations are also performed for the P. denitrificans aa ₃ oxygen reductase. In both oxygen reductases we find several residues that are proton active (i.e., that display partial protonation) at physiological pH, some of them being redox sensitive (i.e., sensitive to the protein redox state). However, the caa ₃ Tyr256^I is not proton active at physiological pH, in contrast to the aa ₃ Glu278^I which is both proton active at physiological pH and shows a high redox sensitivity. In R. marinus caa ₃ we do not find any other residues in the same protein zone that can have this property. Therefore, there are no putative D-channel residues that are proton active in this oxidase. The protonatable residues of the K-channel are much more functionally conserved in both oxygen reductases than the same type of residues in the D-channel. Two (Tyr262^I and Lys336^I, caa ₃ numbering) out of three protonatable K-channel residues are proton active and redox sensitive in both proteins.     

The reactions of the oxidase and reductases ofParacoccus denitrificans with cytochromesc

Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec ₅₅₂ than with either solubleParacoccus c ₅₅₀ or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.     

Direct monitoring of the electron pool effect of cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>3</Subscript> by highly sensitive EQCM measurements

Cytochrome c₃ from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c₃ with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c₃ molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c₃ is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c₃ to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c₃ and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c₃ stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c₃ facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c₃ donates the accepted electrons to the viologen with difficulty.     

The pathway of electron transfer in the dimeric QH2: Cytochromec oxidoreductase

The experimental data currently available suggest that QH₂: cytochromec oxidoreductase functions according to a Q-cycle type of mechanism. The molecular weight of the enzyme in a natural or artificial phospholipid bilayer or in solution corresponds to that of a dimer. The pre-steady state kinetics of reduction of the prosthetic groups indicate that the enzyme is functionally dimeric. A double Q cycle is proposed, describing the pathway of electron transfer in the dimeric QH₂: cytochromec oxidoreductase. According to this scheme, the two monomeric halves of the enzyme act in a cooperative fashion to complete the catalytic cycle. It is proposed that high-potential cytochromeb-562 and low-potential cytochromeb-562 act cooperatively, viz. as a functional pair, in the antimycin-sensitive reduction of ubiquinone to ubiquinol.     

Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb ₅. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb ₅, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb ₅, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.Abbreviations CMP cytidine 5-monophosphate - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - DTT dithiothreitol     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.     

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

A further investigation of the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>–cytochrome<Emphasis Type="Italic"> c</Emphasis> complex

The interaction of reduced rabbit cytochrome b₅ with reduced yeast iso-1 cytochrome c has been studied through the analysis of ¹H–¹⁵N HSQC spectra, of ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b₅ has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b₅.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics     

A proposed pathway of proton translocation through thebc complexes of mitochondria and chloroplasts

The cytochromebc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50–70% with minimal effect on electron transfer in the cytochromebc ₁ and cytochromebf complexes reconstituted into liposomes. Subsequent binding studies with cytochromebc ₁ and cytochromebf complexes indicate that DCCD specifically binds to the subunitb and subunitb ₆, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochromeb and aspartate 155 or glutamate 166 of spinach cytochromeb ₆. Moreover, similar inhibition of proton translocating activity and binding to cytochromeb and cytochromeb ₆ were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochromeb and cytochromeb ₆ is localized near the surface of the membrane but shielded from the external medium.     

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb 5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb ₅ performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb ₅ is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.     

Thebc 1 complexes ofRhodobacter sphaeroides andRhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochromec oxidoreductase (bc ₁ complex). In bothRhodobacter sphaeroides andRhodobacter capsulatus, thebc ₁ complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because thebc ₁ complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochromeb subunit, in the Rieske iron-sulfur subunit, and in the cytochromec ₁ subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q_o) site or the quinol reductase (Q_i) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.     

Implications of cytochromeb 6/f location for thylakoidal electron transport

The cytochromeb ₆/f complex of higher plant chloroplasts is uniformly distributed throughout both appressed and nonappressed thylakoids, in contrast to photosystem II and photosystem I, the other major membrane protein complexes involved in electron transport. We discuss how this distribution is likely to affect interactions of the cytochromeb ₆/f complex with other electron transport components because of the resulting local stoichiometries, and how these may affect the regulation of electron transport.     

Effect of ubiquinone extraction on the reaction of the mitochondrialbc 1 complex with ferricyanide

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc ₁ complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.     

What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: Cytochromec oxidoreductase?

The Q cycle mechanism of thebc ₁ complex requires two quinone reaction centers, the hydroquinone oxidation (Q_P) and the quinone reduction (Q_N) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of Q_P site inhibitors to thebc ₁ complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutantbc ₁ complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochromeb and parts of the iron-sulfur protein. At least two different binding sites for Q_P site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochromeb, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the Q_P pocket is proposed.     

Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Functional characterization of the lesion in the ubiquinol: Cytochromec oxidoreductase complex isolated from the nonphotosynthetic strain R126 ofRhodobacter capsulatus

The cytochromebc ₁ complexes from the nonphotosynthetic strain R126 ofRhodobacter capsulatus and from its revertant MR126 were purified. Between both preparations, no difference could be observed in the stoichiometries of the cytochromes, in their spectral properties, and in their midpoint redox potentials. Both also showed identical polypeptide patterns after electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate. The ubiquinol: cytochromec oxidoreductase activity was strongly inhibited in the complex from the mutant compared to the one from the revertant. So was the oxidant-induced extra reduction of cytochromeb. Both preparations, however, showed an antimycin-induced red shift of cytochromeb, as well as antimycin-sensitive reduction of cytochromeb by ubiquinol. In accordance with a preceding study of chromatophores (Robertsonet al. (1986).J. Biol. Chem. 261, 584–591), it is concluded that the mutation affects specifically the ubiquinol oxidizing site, leaving the ubiquinol reducing site unchanged.     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.