The effects of several nucleotides on the molecular state and catalytic activity of Thiobacillus novellus cytochrome c oxidase. ATP affects the oxidase uniquely.

The catalytic activity and molecular aspects of Thiobacillus novellus cytpchrome c oxidase were affected by ATP. The steady-state kinetics in the oxidation of ferrocytochrome c by the oxidase varied with the presence or absence of ATP; the [S]-v curve of the reaction was sigmoid in the absence of ATP whereas it was a Michaelis-Menten-type hyperbola in the presence of 700 microM ATP. The oxidase was a dimer of the minimal structural subunit consisting of one molecule each of two subunits in the presence of Tween 20 and in the absence of ATP. The dimer dissociated into monomers in the presence of 700 microM ATP. The trough at 452 nm seen in the second derivative absorption spectrum of the CO compound of the oxidase in the absence of ATP, a characteristic of the cytochrome a component of cytochrome aa3, dissappeared in the presence of 700 microM ATP. However, ADP, AMP, GTP, CTP and UTP had little affect on both the [S]-v curve and the molecular mass of the oxidase when used in place of ATP.     

Localization of Cu and heme a on cytochrome c oxidase polypeptides.

After mild dissociation of cytochrome $\text{c}$ oxidase protomers, and polyacrylamide gel electrophoresis, copper was found predominantly in polypeptides of Bands V (m.w. 12,100) and VII (m.w. 3,400), and heme a predominantly in polypeptides of Bands I (m.w. 35,300) and II (m.w. 21,000). Some copper was found in Band II – III, and heme a in Band V.     

Thiobacillus ferrooxidans cytochrome c oxidase: purification, and molecular and enzymatic features.

Cytochrome c oxidase from Thiobacillus ferrooxidans was purified to homogeneity and some of its properties were studied. The oxidase was solubilized with n-octyl-beta-D-thioglucoside (OTG) under acidic conditions (pH 4.0) and purified by one step of ion-exchange chromatography with a CM-Toyopearl column. The absorption spectrum of the oxidase showed peaks at 420 and 595 nm in the oxidized form and at 440 and 595 nm in the reduced form. Its CO compound showed a novel absorption spectrum; a double-peaked gamma band appeared at 429 and 438 nm. The oxidase seemed to have CuA-like copper atom from its ESR and near-infrared spectra. The oxidase molecule consisted of three polypeptides with molecular weights of 53,000, 22,000, and 17,000, respectively, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme in a solution containing detergents was estimated to be 169,000 on the basis of the results obtained by gel filtration, while the molecular weight per heme alpha was estimated to be 83,700. The copper content of the oxidase was 1.01 g atom per mol of heme alpha. Therefore, the cytochrome seemed to contain one molecule of heme alpha and one atom of copper in the minimal structural unit consisting of one molecule each of the three subunits, and to occur as a dimer of the unit in the solution. The oxidase oxidized ferrocytochrome c-552 of the bacterium, and the optimal pH of the reaction was 3.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Electron spin relaxation of CuA and cytochrome a in cytochrome c oxidase. Comparison to heme, copper, and sulfur radical complexes

The method of continuous saturation has been used to measure the electron spin relaxation parameter T1T2 at temperatures between 10 and 50 K for a variety of S = 1/2 species including: CuA and cytochrome a of cytochrome c oxidase, the type 1 copper in several blue copper proteins, the type 2 copper in laccase, inorganic Cu(II) complexes, sulfur radicals, and low spin heme proteins. The temperature dependence and the magnitude of T1T2 for all of the species examined are accounted for by assuming that the Van Vleck Raman process dominates the electron spin-lattice relaxation. Over the entire temperature range examined, the relaxation of the type 1 coppers in six to seven times faster than that of type 2 copper, inorganic copper, and sulfur radicals, in spite of the similar g-anisotropies of these species. This result may indicate that the coupling of the phonon bath to the spin center is more effective in type 1 coppers than in the other complexes studied. The relaxation of CuA of cytochrome oxidase exhibits an unusual temperature dependence relative to the other copper complexes studied, suggesting that the protein environment of this center is different from that of the other copper centers studied and/or that CuA is influenced by a magnetic dipolar interaction with another, faster-relaxing paramagnetic site in the enzyme. A comparison of the saturation characteristics of the CuA EPR signal in native and partially reduced CO complexes of the enzyme also suggests the existence of such an interaction. The implications of these results with respect to the disposition of the metal centers in cytochrome oxidase are discussed.     

Mitochondrial copper metabolism and delivery to cytochrome c oxidase

Metals are essential elements of all living organisms. Among them, copper is required for a multiplicity of functions including mitochondrial oxidative phosphorylation and protection against oxidative stress. Here we will focus on describing the pathways involved in the delivery of copper to cytochrome c oxidase (COX), a mitochondrial metalloenzyme acting as the terminal enzyme of the mitochondrial respiratory chain. The catalytic core of COX is formed by three mitochondrially-encoded subunits and contains three copper atoms. Two copper atoms bound to subunit 2 constitute the Cu(A) site, the primary acceptor of electrons from ferrocytochrome c. The third copper, Cu(B), is associated with the high-spin heme a(3) group of subunit 1. Recent studies, mostly performed in the yeast Saccharomyces cerevisiae, have provided new clues about 1) the source of the copper used for COX metallation; 2) the roles of Sco1p and Cox11p, the proteins involved in the direct delivery of copper to the Cu(A) and Cu(B) sites, respectively; 3) the action mechanism of Cox17p, a copper chaperone that provides copper to Sco1p and Cox11p; 4) the existence of at least four Cox17p homologues carrying a similar twin CX(9)C domain suggestive of metal binding, Cox19p, Cox23p, Pet191p and Cmc1p, that could be part of the same pathway; and 5) the presence of a disulfide relay system in the intermembrane space of mitochondria that mediates import of proteins with conserved cysteines motifs such as the CX(9)C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis.     

Protonation states of the catalytic intermediates of cytochrome c oxidase.

Protonation changes accompanying conversion of oxidised (O state) cytochrome c oxidase to the 2-electron-reduced P state, and 3-electron-reduced F state at pH 8.0 have been measured. It was found that 2 and 3 protons, respectively, were taken up. The fourth proton required for the reduction of O2 to H2O must therefore be consumed in the remaining F----O portion of the catalytic cycle.     

Location of heme a on subunits I and II and copper on subunit II of cytochrome c oxidase

A systemic study has been made of copper and heme a binding to subunits of beef heart cytochrome c oxidase. Copper and heme a were readily mobilized by ionic detergents, high ionic strengths, temperatures above 0 degrees C, thiol compounds, and gel-bound peroxides and free radicals when the subunits of the oxidase were dissociated from one another during polyacrylamide gel electrophoresis. Most subunits showed some affinity for heme a and copper under these conditions. However, in the presence of specific mixtures of ionic and nonionic detergents (e.g. 0.1% sodium dodecyl sulfate, 0.025% Triton X-100) at temperatures below 0 degrees C and in buffers of low ionic strength using 10 to 12% polyacrylamide gels preelectrophoresed for 3 days with thioglycolate, about 90% of the Cu was found on subunit II (Mr = 24,100), and heme a was found in equal amounts of subunits I (Mr = 35,800) and II. The oxidized-reduced and reduced-CO absorption spectra of these subunits resembled those of cytochrome c oxidase. It appears probable that in the native enzyme, subunit I contains heme a and subunit II contains copper and heme a. A relationship of mammalian cytochrome c oxidase to the two-subunit microbial cytochrome oxidase systems appears to exist.     

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.     

Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin which assembly is intricate and highly regulated. The COX catalytic core is formed by three mitochondrial DNA encoded subunits, Cox1, Cox2 and Cox3, conserved in the bacterial enzyme. Their biogenesis requires the action of messenger-specific and subunit-specific factors which facilitate the synthesis, membrane insertion, maturation or assembly of the core subunits. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to identify these ancillary factors. Here we review the current state of knowledge of the biogenesis and assembly of the eukaryotic COX catalytic core and discuss the degree of conservation of the players and mechanisms operating from yeast to human. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

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.     

Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase.

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.