Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at ?40°C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichiometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method.$\text{→}\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed $\text{→}\text{H}{}^{\mathrm{+}}\text{O}$ with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygenand ferricyanide pulses, with endogenous substrates or added methanol as a substrte, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH^? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.     

Ferrocyanide as electron donor to cytochrome aa3. Cytochrome c requirement for oxygen uptake

1. In the absence of cytochrome c, ferrocyanide or ferrous sulphate reduces cytochrome c oxidase (EC 1.9.3.1), but no continuous oxygen uptake ensues, as it does with N,N,N′,N′-Tetramethyl-p-phenylenediamine or reduced phenazine methosulphate as reductants, unless a substoichiometric amount of cytochrome c or an excess of clupein is present. Cytochrome c cannot be replaced by porphyrin cytochrome c.2. Cytochrome c, porphyrin cytochrome c and clupein all stimulate the reduction of cytochrome aa₃ by ferrocyanide.3. A model is proposed to explain these findings in which a high-affinity site for cytochrome c on the oxidase regulates the access of hydrophilic electron donors to a low-affinity site, and reduction via the high-affinity site is required for continuous oxygen uptake.4. Furthermore, it is shown that upon reaction of oxidase with ferrocyanide, cyano-oxidase is formed.     

The participation of cytochromes in the process of nitrate respiration in Klebsiella (Aerobacter) aerogenes

1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a₁, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a₁, d and o.     

Effect of cardiolipin on the enzymatic activity of Nitrobacter agilis cytochrome c oxidase

Effects of cardiolipin on the reaction rates of Nitrobacter agilis cytochrome c oxidase with cytochrome c were studied at various concentrations of phosphate buffer. Cardiolipin stimulated greatly the oxidation by the enzyme of horse and yeast ferrocytochromes c, especially at higher ionic strengths. However, the oxidation by the enzyme of N. agilis ferrocytochrome c-550, the physiological electron donor for the oxidase, was not accelerated by addition of cardiolipin. Analysis of the lipid compositions showed that neither the cell membranes of N. agilis nor the enzyme preparation contained cardiolipin. These results suggest that cardiolipin is not necessary for the reaction of N. agilis cytochrome c oxidase with N. agilis cytochrome c-550. On the basis of these results, the difference in the reactivity with cytochrome c of cytochrome c oxidase between the bacterial and mitochondrial enzymes is discussed.     

Functional and Structural Comparisons between Prokaryotic and Eukaryotic aa3-Type Cytochrome c Oxidases from an Evolutionary Point of View

The specificities for cytochrome c of the aa3-type cytochromec oxidase were studied with enzymes derived from Thiobacillusnovellas, Nitrobacter agilis, Paracoccus denitrificans and thecow in reaction with the cytochromes c from 5 prokaryotes and7 eukaryotes. The T. novellus enzyme reacted most rapidly withthe cytochromes c of Candida krusei, tuna and bonito as wellas T. novellus cytochrome c; the specificity for cytochromec of the N. agilis enzyme was similar to that of the T. novellusenzyme. The bovine enzyme reacted rapidly with all the eukaryoticcytochromes c tested. The P. denitrificans enzyme showed a specificitysimilar to that of the bovine enzyme, except that it reactedrapidly with P. denitrificans cytochrome c, while the bovineenzyme reacted with it very poorly. All four kinds of enzymesshowed an extremely limited reaction with Pseudomonas aeruginosacytochrome c. The amino acid composition of subunit I of the N. agilis enzymeresembled that of the bovine enzyme, while the compositionsof their subunits II were different. On the basis of these results,an evolutionary relationship between bacterial and eukaryoticenzymes was discussed. (Received May 21, 1981; Accepted August 20, 1981)     

Association of Cytochrome c with Membrane-Bound Cytochrome c Oxidase Proceeds Parallel to the Membrane Rather Than in Bulk Solution

Electron transfer between the water-soluble cytochrome c and the integral membrane protein cytochrome c oxidase (COX) is the terminal reaction in the respiratory chain. The first step in this reaction is the diffusional association of cytochrome c toward COX, and it is still not completely clear whether cytochrome c diffuses in the bulk solution while encountering COX, or whether it prefers to diffuse laterally on the membrane surface. This is a rather crucial question, since in the latter case the association would be strongly dependent on the lipid composition and the presence of additional membrane proteins. We applied Brownian dynamics simulations to investigate the effect of an atomistically modeled dipalmitoyl phosphatidylcholine membrane on the association behavior of cytochrome c toward COX from Paracoccus denitrificans. We studied the negatively charged, physiological electron-transfer partner of COX, cytochrome c₅₅₂, and the positively charged horse-heart cytochrome c. As expected, both cytochrome c species prefer diffusion in bulk solution while associating toward COX embedded in a membrane, where the partial charges of the lipids were switched off, and the corresponding optimal association pathways largely overlap with the association toward fully solvated COX. Remarkably, after switching on the lipid partial charges, both cytochrome c species were strongly attracted by the inhomogeneous charge distribution caused by the zwitterionic lipid headgroups. This effect is particularly enhanced for horse-heart cytochrome c and is stronger at lower ionic strength. We therefore conclude that in the presence of a polar or even a charged membrane, cytochrome c diffuses laterally rather than in three dimensions.     

Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba₃-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa₃-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa₃-type cytochrome c oxidase and the ba₃-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.     

Assembly of a chimeric respiratory chain from bovine heart submitochondrial particles and cytochrome bd terminal oxidase of Escherichia coli

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc₁ (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.     

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+a₃, b-562, b-555, c and d, with three potential oxidases: cytochromes a+a₃, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate-N,N,N′,N′-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.     

The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied.The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor.The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a ‘cytochrome bd’-type oxidase; a novel cytochrome.     

Exogenous cytochrome c-dependent light-induced membrane potential generation in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores associated with a planar phospholipid macromembrane by bivalent cations in the presence of quinone, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and ascorbate generate a transmembrane electrical potential difference in the light. Photoelectrical activity is also observed if chromatophores are preincubated with cytochrome c; the maximum values of responses are reached upon subsequent addition of ascorbate and menadion in the absence of bivalent cations and TMPD. The cytochrome c-dependent responses of the illuminated chromatophores are inhibited by Ca²⁺ and prevented by quinones. The possibility of cytochrome c (c₂) translocation across the chromatophore membrane and the mechanism of charge transfer across the planar phospholipid membrane are discussed.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

Separation of respiratory reactions in Rhodospirillum rubrum: Inhibition studies with 2-hydroxydiphenyl

1. Respiration of chemotrophically and phototrophically grown Rhodospirillum rubrum is inhibited by 2-hydroxydiphenyl.2. Membrane-bound NADH oxidase and NADH: cytochrome c reductase are inhibited also. The inhibitor constant for both reactions (K_i) is 0.075±0.012 mM. NADH dehydrogenase is not inhibited significantly.3. The inhibition of succinate:cytochrome c reductase is associated for chemotrophic membranes with K_i = 0.22±0.03 mM and for phototrophic membranes with K_i = 0.49±0.09 mM. Succinate dehydrogenase is not affected by 2-hydroxydiphenyl.4. Cytochrome oxidase is inhibited only slightly.5. While NADH-dependent reactions in both phototrophic and chemotrophic membranes are inhibited maximally more than 95%, succinate-dependent reactions can be inhibited more than 95% only in chemotrophic membranes. In photo-trophic membranes the maximum inhibition of succinate-dependent reactions is about 70%.6. The type of inhibition in both cases 2 and 3 is non-competitive.7. While the reduction of b-type cytochrome is inhibited by 2-hydroxydiphenyl, the degree of ubiquinone reduction is not influenced. The data suggest that the site of inhibition is localized between ubiquinone and cytochrome b.8. Implications of these data for the respiratory electron transport system in R. rubrum are discussed.     

Respiratory components and oxidase activities in Alcaligenes eutrophus

1. Cells of the hydrogen bacterium Alcaligenes eutrophus are broken by gentle lysis using lysozyme treatment in hypertonic sucrose followed by osmotic shock. By this method, 93% of the in vivo activity of the H₂ oxidase is recovered and the ATPase remains particle bound. In contrast, cell disruption in a French pressure cell diminishes the in vivo activity of the H₂ oxidase by 50% and solubilizes the bulk of the ATPase.2. The bacterium contains a periplasmic cytochrome c with bands at 418, 521 and 550 nm (difference spectrum). In addition to cytochrome aa₃, b-560, c-553 and o, low temperature difference spectra of membranes show the presence of two further cytochromes (shoulders at 551 and 553 nm).3. The unsupplemented membrane fraction catalyses the oxidation of hydrogen, NADH, NADPH, succinate, formate and endogenous substrate (NAD linked) at rates 2–3-fold higher than membranes obtained from cells disrupted in a French pressure cell. With the exception of the H₂ oxidase all oxidase activities in lysozyme membranes are sensitive to carbonylcyanide m-chlorophenylhydrazone (20–100% stimulation of oxygen uptake).4. The cytoplasmic fraction contains a B-type cytochrome with absorption maxima at 436 and 560 nm, capable of combining with CO; it contains non-covalently bound protohaem. In alkaline solutions a spectral transition to the haemochrome type with bands at 423, 526 and 556 nm occurs. The addition of NADH to an aerobic suspension of this cytochrome elicits new absorption maxima at 418, 545 and 577 nm (difference spectrum), which are believed to represent an oxygenated form of the reduced cytochrome.     

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

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

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Regulation of respiration in paracoccus denitrificans: the dependence on redox state of cytochrome c and [ATP]/[ADP][Pi].

The respiratory rates of Paracoccus denitrificans cells, membrane fragments, and detergent-solubilized, ammonium sulfate-precipitated membrane fractions were measured with NADH and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine as the substrates. It was found that the turnover numbers for cytochrome c were the same in the three preparations giving maximum values of approximately 300 s^?1 at 100% reduction of cytochrome c. NADH was rapidly oxidized in the detergent-solubilized, ammonium sulfate-precipitated membrane fraction which contained tightly bound cytochrome c. It is suggested that tight binding of cytochrome c in P. denitrificans does not impair its electron transport activity. The respiration of intact cells was dependent on the redox state of cytochrome c over a wide range of cytochrome c reduction, on the intracellular [ATP]/[ADP][P_i] and on the pH of the suspending medium. A conclusion is drawn that the basic principle(s) underlying regulation of cellular respiration is the same in the prokaryotic P. denitrificans and in mitochondria-containing eukaryotic organisms.     

Oxidation of exogenous c-type cytochromes by intact spheroplasts of Anacystis nidulans

Intact spheroplasts of the cyanobacterium Anacystis nidulans were found to oxidize reduced c-type cytochromes derived from horse heart, tuna, Saccharomyces oviformis, Candida krusei, Rhodocyclus purpureus, Rhodopseudomonas plustris and Paracoccus denitrificans with characteristics similar to those observed with isolated membranes. Rates of cytochrome c oxidation by the spheroplasts were only 10% of those measured with isolated membranes in which thylakoid-bound cytochrome oxidase contributes to the overall rates. Small amounts of an endogenous c-type cytochrome were released upon lysozyme treatment of the cells. The results appear to indicate the presence of cytochrome oxidase in the cytoplasmic membrane of A. nidulans.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - cyt cytochrome(s)