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.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

The iron(III)-adriamycin complex inhibits cytochrome c oxidase before its inactivation.

Cytochrome c oxidase was found to be competitively inhibited by a complex formed between Fe3+ and the cardiotoxic antitumour drug adriamycin (doxorubicin) with an inhibition constant, Ki, of 12 microM. This competitive inhibition precedes the slower Fe3+-adriamycin induced inactivation of cytochrome c oxidase. In strong contrast with this result, free adriamycin was not observed to either inhibit or inactivate cytochrome c oxidase (Ki greater than 3 mM). Since, typically, polycations are known to inhibit cytochrome c oxidase, the competitive inhibition displayed by the Fe3+-adriamycin complex may also result from its polycationic character. Cytochrome c oxidase was also inhibited by pentan-1-ol (Ki 13 mM), and kinetic studies carried out in the presence of both inhibitors demonstrated that the Fe3+-adriamycin complex and pentan-1-ol are mutually exclusive inhibitors of cytochrome c oxidase. The inhibitor pentan-1-ol was also effective in preventing the slow inactivation of cytochrome c oxidase induced by Fe3+-adriamycin, presumably by blocking its binding to the enzyme. It is postulated that the slow inactivation of cytochrome c oxidase occurs when reactive radical species are produced while the Fe3+-adriamycin is complexed to cytochrome c oxidase in an enzyme-inhibitor complex. The Fe3+-adriamycin-induced inactivation of cytochrome c oxidase may be, in part, responsible for the cardiotoxicity of adriamycin.     

Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry

The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.     

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.     

Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.     

Cytochrome c1 complexes.

Cytochrome c1 forms an active complex with cytochrome c as previously reported (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36). It also forms a complex with cytochrome oxidase with heme ratio of 1:1. This cytochrome c1.oxidase complex has been purified by ammonium sulfate fractionation and is stable in media of high ionic strength (greater than 0.1 M) but dissociates as the pH deviates from neutral. The purified cytochrome c1 aggregates to an oligomer, presumably a pentamer. No agent has been found to depolymerize isolated c1 without denaturation. However, in the cytochrome c1.oxidase complex, these two cytochromes apparently were depolymerized to form smaller aggregates, if not monomeric units, as judged by sedimentation behavior. Cytochrome c1 also forms a ternary complex with cytochrome c and oxidase in the heme ratio of 1:1:1. This complex can be prepared by any of the following four methods: (i) c1 + c + oxidase: (ii) c1.c complex + oxidase; (iii) c1 + c.oxidase complex: or (iv) c + c1.oxidase complex. The mode of formation of these complexes is all from pure protein-protein interactions. Cytochrome c1 is also incorporated into phospholipid vesicles and these vesicles show about 200 molecules of phospholipid/cytochrome c1 in terms of heme. The spectrophotometric, circular dichroic, sedimentation behavior and enzymic properties of these complexes have been investigated.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)     

The photoreactivity of the copper-NO complexes in cytochrome c oxidase and in other copper-containing proteins

The complexes of NO with CuB of cytochrome c oxidase in which cytochrome a3 may or may not be ligated to cyanide or fluoride are photodissociable. NO does not appear to react with CuB in complexes of cytochrome c oxidase in which sulphide or mercaptans are ligated to the haem iron of cytochrome a3. A comparison is made between the photoreactivity of the complexes of NO with cytochrome c oxidase and those with ceruloplasmin, ascorbate oxidase, and haemocyanin. It is shown that the photoreactivity of CuB 2+.NO in cytochrome c oxidase is not unique for this enzyme, but may also be observed in the complexes of NO with type-1 copper-containing enzymes. This would suggest that the ligation of CuB in cytochrome c oxidase shows some similarity to type-1 copper in blue oxidases.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen.

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen is measured by transient absorption spectroscopy. The oxygen reaction is initiated by photolytic removal of CO from cytochrome oxidase, using a flash-pumped dye laser. The subsequent reaction of the cytochrome c-cytochrome oxidase complex with oxygen is reported at 550, 605, 744, and 830 nm at different cytochrome c:cytochrome oxidase ratios and different oxygen concentrations. In the absence of cytochrome c the time course of the reaction of the oxidase is well described by a triple exponential process at any of the measured wavelengths. The three processes are well resolved at high O2 levels (i.e. greater than 200 microM), where they reach first-order rate limits of 2.4 x 10(4), 7.5 x 10(3), and 650 s-1. When cytochrome c is added the oxidation of cytochrome a and one of the redox active cooper centers (CuA) are interrupted. The maximal effect of cytochrome c on the oxidation of the oxidase occurs at a c:aa3 ratio of 1. Cytochrome c reacts in a biphasic process with rates of up to 7 x 10(3) and 550 s-1 at high oxygen. The fast phase takes up 60% of the process, and this is independent of the cytochrome c:cytochrome oxidase ratio. The results are discussed in the context of a model in which electron entry into cytochrome oxidase from cytochrome c is via CuA, and cytochrome a functions to mediate electron transfer from CuA to the oxygen binding site. The role of CuA as initial electron acceptor in cytochrome c oxidase is related to its physical proximity to cytochrome c is the cytochrome c-cytochrome oxidase complex.     

Calmodulin stimulation of adenylate cyclase of intestinal epithelium

The effect of dicyclohexylcarbodiimide (DCCD) on the proton pumping two-subunit cytochrome c oxidase from Paracoccus denitrificans was investigated. Purified Paracoccus oxidase was reconstituted into phospholipid vesicles by cholate dialysis. Following incubation with increasing amounts of DCCD, proton ejection was recorded in response to reductant pulses with reduced cytochrome c. Concentrations of DCCD which greatly reduced proton pumping by bovine cytochrome c oxidase used as a control were found to exert only a minor effect on proton translocation by Paracoccus oxidase. Similarly, incubation of the bacterial enzyme with [14C]DCCD failed to reveal the specific covalent interaction previously demonstrated to occur with bovine cytochrome c oxidase, and here also shown for the oxidase of yeast. Thus, Paracoccus oxidase differs in its interaction with DCCD from the functionally analogous eukaryotic enzymes.     

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.     

Isolation and characterization of COX12, the nuclear gene for a previously unrecognized subunit of Saccharomyces cerevisiae cytochrome c oxidase.

We have cloned and sequenced COX12, the nuclear gene for subunit VIb of Saccharomyces cerevisiae cytochrome c oxidase. This subunit, which was previously not found in cytochrome c oxidase purified from S. cerevisiae, has a deduced amino acid sequence which is 41% identical to the sequences of subunits VIb of bovine and human cytochrome c oxidases. The chromosomal copy of COX12 was replaced with a plasmid-derived copy of COX12, in which the coding region for the suspected cytochrome oxidase subunit was replaced with the yeast URA3 gene. The resulting Ura+ deletion strain grew poorly at room temperature and was unable to grow at 37 degrees C on ethanol/glycerol medium, whereas growth was normal at both temperatures on dextrose. This temperature-dependent, petite phenotype of the deletion strain was complemented to wild-type growth with a single copy plasmid carrying COX12. Cytochrome c oxidase activity in mitochondrial membranes from the cox12 deletion strain is decreased to 5-15% of that in membranes from the wild-type parent, and this activity is restored to normal when the cox12 deletion strain is complemented by the plasmid-borne COX12. Optical spectra of mitochondrial membranes from the cox12 deletion strain revealed that optically detectable cytochrome c oxidase is assembled at room temperature and at 37 degrees C, although the heme a + a3 absorption is diminished approximately 50%. The N-terminal amino acid sequence of the protein encoded by COX12 is identical to the N-terminal sequence of a subunit found in yeast cytochrome c oxidase purified by a new procedure (Taanman, J.-W., and Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485). We conclude that COX12 encodes a subunit of yeast cytochrome c oxidase which is essential during assembly for full cytochrome c oxidase activity but apparently can be removed after the oxidase is assembled, with retention of oxidase activity. This is the first instance in which deletion of a subunit of cytochrome c oxidase results in assembly of optically detectable cytochrome c oxidase but having markedly diminished activity.     

Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Ionic-strength-dependence of the oxidation of native and pyridoxal 5''-phosphate-modified cytochromes c by cytochrome c oxidase.

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.     

Cytochrome c-cytochrome oxidase interaction at subzero temperatures.

Cytochrome oxidase forms two distinctive compounds with oxygen at --105 and --90 degrees C, one appears to be oxycytochrome oxidase (Compound A) and the other peroxycytochrome oxidase (Compound B). The functional role of compound B in the oxidation of cytochrome c has been examined in a variety of mitochondrial preparations. The rate and the extent of the reaction have been found to be dependent upon the presence of a fluid phase in the vicinity of the site of the reaction of cytochrome c and cytochrome oxidase. The kinetics of cytochrome c oxidation and of the slowly reacting component of cytochrome oxidase are found to be linked to one another even in cytochrome c depleted preparations, but under appropriate conditions, especially low temperatures, the oxidation of cytochrome c precedes that of this component of cytochrome oxidase. Based upon the identification of the slowly reacting components of cytochrome oxidase with cytochrome c, various mechanisms are considered which allow cytochrome c to be oxidized without the intervention of cytochrome a at very low temperatures, and tunneling seems an appropriate mechanism.