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.     

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.     

Alkali-induced reduction of the b-cytochromes in purified Complex III from beef heart mitochondria

Approx. 40–50% of the cytochrome b in purified Complex III is reduced by ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine or phenazine methosulfate at neutral pH. The remaining cytochrome b, including cytochrome b-565, is reduced by increasing the pH. The apparent pK for this reduction is between pH 10 and 11, and is more than two pH units higher than a similar alkali-induced transition in Mg-ATP particles. Alkali-induced reduction of cytochrome b occurs concomitantly with the exposure of hydrophobic tyrosine and tryptophan residues to a more hydrophilic environment. The relationship of these findings to the presence of a substrate accessibility barrier in Complex III is discussed.     

The thermodynamic properties of some commonly used oxidation-reduction mediators,inhibitors and dyes,as determined by polarography

The oxidation-reduction midpoint potentials (E_m) of the following compounds have been measured in the range of pH from 3 to 12 by polarography: methyl viologen; benzyl viologen; 2-hydroxy-1,4-naphthoquinone; 2-hydroxy-1,4-anthraquinone; N,N,N′,N′,-tetramethyl-p-phenylenediamine;2,3,5,6-tetramethyl-p-phenylenediamine; phenazine; N-methylphenazonium methosulfate; N-methylphenazonium sulfonate methosulfate; N-ethylphenazonium ethosulfate; pyocyanine; neutral red; safranin; phenol red; chlorophenol red; cresol red; bromocresol purple; 2,5-dibromo-3-methyl-6-isopropylbenzoquinone and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Many of these previously assumed to have a simple behavior in this range have proven to be rather more complicated, and several anomalous observations have been reconciled.     

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.     

N,N,N′,N′-tetramethyl-p-phenylenediamine initiates the appearance of a well-resolved I peak in the kinetics of chlorophyll fluorescence rise in isolated thylakoids

Addition of N,N,N′,N′-tetramethyl-p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J–I–P phase of fluorescence rise and greatly retarded the I–P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence (F_v) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles

1. Beef heart mitochondria have a cytochrome c₁ : c : aa₃ ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s^?1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s^?1, at 28° – 30°C, pH 7.4.2. Ascorbate plus N,N,N′,N′-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c₁ and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively.3. Cytochrome c₁ is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+N,N,N′,N′-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c₁ and c in the aerobic steady state, with a rate constant for the c₁ → c reduction step greater than 10³ s^?1.4. The greater apparent response of the $\text{c}\text{aa}{}_3$ electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the $\text{c}{}_1\text{c}$ step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa₃ units in situ. Cytochrome c₁ can either reduce the cytochrome c-cytochrome aa₃ complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Properties of Ascaris muscle mitochondria. I. Cytochromes

1. The cytochrome system in Ascaris muscle mitochondria was further characterized using purer preparations.2. Difference spectra (at 22 °C and ?196 °C) of the mitochondrial preparations using succinate and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine show that Ascaris muscle mitochondria contain cytochromes c₁, c and aa₃, and also at least three b-type cytochromes. The b-type cytochrome is the predominant component.3. Cytochrome c and Ascaris cytochrome b-560 can be extracted from the mitochondrial preparations with 150 mM KCl, leaving the membrane-bound cytochromes c₁, b and aa₃ in the KCl residue.     

Heterogeneity in photosystem I. Evidence from cation-induced decrease in Photosystem I electron transport

The rate of electron transfer through Photosystem I (reduced 2,6-dichlorophenol indophenol (DCIPH₂ → methylviologen) in a low-salt thylakoid suspension is inhibited by Mg²⁺ both under light-limited and the light-saturated conditions, the magnitude of inhibition being the same. The 2,6-dichlorophenol indophenol (DCIP) concentration dependence of the light-saturated rate in the presence and in the absence of Mg²⁺ shows that the overall rate constant of the photoreaction is not altered by Mg²⁺. With N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethylphenylenediamine as electron donor only the light-limited rate, not the light-saturated rate, is inhibited by Mg²⁺ and the magnitude of inhibition is the same as with DCIP as donor. The results are interpreted in terms of heterogeneous Photosystem I, consisting of two types, PS I-A and PS I-B, where PS I-A is involved in cation-regulation of excitation energy distribution and becomes unavailable for DCIPH₂ → methyl viologen photoelectron transfer in the presence of Mg²⁺.     

Rate-limiting step in oxidation of physiological and artificial reductants by Azotobacter vinelandii membrane vesicles.

The respiration of Azotobacter vinelandii membrane vesicles was investigated in order to determine the partial rates of electron fluxes at each segment of its branched respiratory chain. It is concluded that under physiological conditions only 20 to 30% of the total flux is carried through the c₄, c₅ → a₁,o chain. Steady state analysis indicates that the limited capacity of the chain is due to the slow rate of oxidation of the cytochromes c by the a₁,o oxidases. This rate-limiting step is bypassed by the artificial electron donors, ascorbate-2,6-dichlorophenol indophenol and ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine, which directly reduce the highly active a₁,o oxidases. During the oxidation of ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine by the membrane vesicles, an accumulation of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine occurs. Such accumulation of positively charged molecules should lead to a generation of a membrane potential. This fact and previous data concerning coupling site III of A. vinelandii are discussed.     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature

Light-induced absorbance changes were measured at temperatures between ?30 and ?55 °C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the “primary” to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870⁺ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870⁺.A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artificial donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.     

Structural studies on the cytochrome c oxidase proton pump using a spin-label probe

We report studies in which we have used N-(2,2,6,6-tetramethylpiperidyl-1-oxyl)-N′-cyclohexylcarbodiimide, a spinlabel analogue of N,N′-dicyclohexylcarbodiimide, to investigate the structural aspects of the cytochrome c oxidase proton pump. We establish that the spin label binds to the reconstituted enzyme at the same site as does N,N′-dicyclohexylcarbodiimide, i.e., within subunit III. ESR studies of the bound spin label indicate that its binding site is situated in an apolar region of the enzyme, though close to its surface. The binding of the spin label to the free oxidase is different from that with the reconsituted enzyme, leading to spin-spin exchange between the bound probe molecules. From this and the fact that N,N′-dicyclohexylcarbodiimide binds to subunits III and IV in the free oxidase, we conclude that these two subunits are at the most 20 Å apart.     

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H₂O₂, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H₂O₂. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the $\text{O}{}_2\text{NADPH}\text{-supported}$ oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.     

Properties of rat liver mitochondria with intermembrane Cytochrome c.

When rat liver mitochondria were suspended in 0.15 m KCl, the cytochrome c appeared to be solubilized from the binding site on the outside of the inner membrane and trapped in the intermembrane space. When the outer membrane of these mitochondria was disrupted with digitonin at a digitonin concentration of 0.15 mg/mg of protein, the solubilized cytochrome c could be released from mitochondria along with adenylate kinase. When mitochondria were suspended in 0.15 m KCl instead of 0.33 m sucrose, the $\text{ADP}\text{O}$ ratio observed with succinate, β-hydroxybutyrate, malate + pyruvate or glutamate as substrates was little affected. A number of cycles of State 4-State 3-State 4 with ADP was observed. The respiratory control ratios, however, were decreased, particularly when glutamate was used as the substrate. Cytochrome c oxidase activity was also decreased to 55% when assayed using ascorbate + N,N,N′,N′-tetramethyl-p-phenylene-diamine (TMPD) as substrates. Suspension of mitochondria in 0.15 m KCl resulted in an enhancement of the very low NADH oxidation by intact mitochondria and a twofold enhancement of sulfite oxidation. Trapped cytochrome c in outer membrane vesicles prepared from untreated and trypsin-treated intact mitochondria was found to be readily reduced by NADH and suggests that some cytochrome b₅ is located on the inner surface of the outer membrane. The enhanced NADH oxidase could therefore reflect the ability of cytochrome c to mediate intermembrane electron transport. The enhanced sulfite oxidase activity was sensitive to cyanide inhibition and coupled to oxidative phosphorylation ($\text{ADP}\text{O}\text{< 1}$) unlike the activity of mitochondria in sucrose medium. These results suggest that free cytochrome c in the intermembrane space can mediate electron transfer between the sulfite oxidase and the inner membrane.     

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans

The reaction of the cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of Paracoccus denitrificans cytoplasmic membranes with the endogenous cytochrome c of the membranes was studied, as well as its interaction with added exogenous cytochrome c from P. denitrificans or bovine heart. The polarographic method was employed, using N,N,N′,N′-tetramethyl-p-phenylenediamine plus ascorbate to reduce the cytochrome c. We found that overall electron transport can proceed maximally while the cytochrome c remains membrane bound; NADH or succinoxidase activities were not inhibited by the addition of substances which bind the P. denitrificans cytochrome c strongly. In contrast to our observations with the spectrophotometric method (Smith, L., Davies, H.C. and Nava, M.E. (1976) Biochemistry 15, 5827–5831), in the polarographic assays the membrane-bound oxidase reacts with about equal rapidity with exogenous bovine and P. denitrificans cytochromes c. The reaction of the oxidase with the endogenous cytochrome c proceeds at high rates and preferentially to that with exogenous cytochrome c; the reaction with the latter, but not the former is inhibited by positively charged poly(l-lysine). The cytochrome c and the oxidase appear to be very closely associated on the membrane.     

Two electrogenic mechanisms contributing to the 560 nm absorption changes in intact Bryopsis chloroplasts

Light -induced absorbance changes at 560 nm in dark-adapted intact chloroplasts of the green alga, Bryopsis maxima were studied in the time range of 200 ms. The initial rise of the 560 nm signals constists of two major components which are both electrochromic absorbance changes of the carotenoids, sipnonein and/or siphonaxanthin, but different in mechanisms of the field formation.The first component (component S) is related to electron transport since it was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and showed a light-intensity dependence similar to that of electron transport in chloroplasts. In the presence of DCMU, component S could be restored on addition of proton-transporting electron donors such as reduced 2,6-dichlorophenol indophenol and phenazine methosulfate, but not on addition of N,N,N′,N′-tetramethyl-p-phenylenediamine which does not carry protons with electrons (Trebst, A. (1974) Annu. Rev. Plant Physiol. 25, 423–458). We propose that component S is due to the electric field set up by the proton translocation across the thylakoid membrane.The second component (component R) was resistant to DCMU and DBMIB. The light-intensity dependency of component R was similar to that of cytochrome f photooxidation which showed saturation at a relatively low light intensity. The magnitude of component R was markedly reduced by phenylmercuric acetate, suggesting the participation of ferredoxin and ferredoxin-NADP oxidoreductase in the mechanism of the field formation responsible for this component. In the presence of DCMU and phenylmercuric acetate, time courses of the 560 nm changes paralleled those of cytochrome f changes. These results indicate that component R is due to the electric field formed between oxidized cytochrome f and other intersystem electron carriers located in the inner part of the thylakoid membrane and reduced electron acceptors of Photosystem I situated on the membrane surface.The complex natures of the 560 nm changes, as well as the contributions of Photosystems I and II to the absorbance changes, are explained in terms of the two electrogenic mechanisms.     

Comparison of mutagenicity and chemical properties of N-methyl-N′-alkyl-N-nitrosoureas

Thed mutagenic activities of 11 N-methyl-N′-alkyl-N-nitrosoureas were tested on Samonellatyphimurium TA1535 and compared with chemical properties (alkylating activity and decompostion rate). In their relative mutagenicities the N-nitrosoureas that had a cyclic N′-alkyl group showed far more mutagenic activity than those having a chain N′-alkyl group. M(1-A)NU and M(2-A)NU, which had the most bulky N′-alkyl group in this series, exhibited lethal effects at high concentrations. The mutagenicity showed a small positive correlation with decomposition rates but not with alkylating activities on 4-(p-nitrobenzyl_prridine. The highest mutagenicity in this series was observed in N-methyl-N′-cyclobutyl-N-nitrosourea.These results suggest that, in this series of N-methyl-M′-alkyl-N-nitrosoureas, structural differences in the N′-alkyl groups had great significance in mutagenicity.     

The peroxidase nature of cytochrome P-420 utilizing a lipid peroxide substrate

The peroxidase properties of cytochrome P-420, in its “high spin” and “low spin” forms, have been investigated using N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as hydrogen donor, linoleic acid hydroperoxide (LAHPO) as substrate, and a preparation of cytochrome P-420 from hepatic microsomal “P-450 particles” as catalyst. The peroxidase reaction was found to be first order with respect to P-420 concentration and first order with respect to LAHPO concentration. Of the various hydroperoxides tested, only LAHPO was found to serve as a substrate for P-420.