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Applying the metabolic control theory, inhibitor titration studies were carried out on Complex I, III, IV, ATP synthase, ATP/ADP carrier and P(i) carrier of mitochondrial oxidative phosphorylation in normal and regenerating rabbit liver in order to examine the acceleration mechanism of mitochondrial oxidative phosphorylation. In regenerating rabbit liver the rate of state 3 respiration, respiratory control ratio and phosphorylation rate in the presence of mM glutamate, 250 microM ADP and 3 mM inorganic phosphate increased significantly as compared with the control by 73%, 48% and 76%, respectively. The control of the rate of state 3 respiration in normal liver was exerted by Complexes I, IV and steps other than the aforementioned six steps, whose flux control coefficients were 0.317, 0.214 and 0.469, respectively. By contrast, in regenerating liver, the control was more evenly distributed among these steps in oxidative phosphorylation and the possibility is suggested that Complexes I, IV and steps other than the six steps are activated during regeneration. The activation of Complexes I and IV was attributed to their increased activity, since it was not accompanied by an increase in the amount of the enzymes.

A comparison is made of types and distribution of cytochromes and certain ferredoxins (HiPIP) among photosynthetic bacteria. These are subdivided as to the type of reaction center each species is believed to contain. The proteins listed are assumed to be of periplasmic origin. Interrelationships suggested by the comparison are discussed.

The cytochrome d terminal oxidase complex is a heterodimer located in the cytoplasmic membrane of Escherichia coli. Subunit II of the cytochrome d terminal oxidase complex was expressed independently of subunit I of the complex. It was found that the polypeptide is produced and is associated with the cytoplasmic membrane in the absence of subunit I, and is not associated with any of the three cytochrome components of the complex. Oxidase activity and heme binding are restored when the subunit I is expressed in the same cells using a second compatible plasmid. It has been previously demonstrated that subunit I, expressed in the absence of subunit II, contains cytochrome b-558, one of the three heme prosthetic groups found in the oxidase. Association of the two other heme moieties, cytochromes b-595 and d, apparently requires the association of the two subunits, and must be a late step in the assembly of the membrane-bound protein. It was also shown that under heme-deficient conditions, the two polypeptide subunits are expressed and are associated with the cytoplasmic membrane.

The cytochrome oxidase concentration and content of rat brain during development was measured using a simple new assay for cytochrome a. The cytochrome oxidase concentration increased from 1.2 nmol/g wet wt. of brain at birth to about 5.5 nmol/g in the adult, most of the change occurring between 5 and 25 days after birth.

An inner mitochondrial membrane fraction was prepared from porcine corpus luteum. The concentrations of the respiratory cytochromes, cytochrome P-450scc, cholesterol, ubiquinone, cardiolipin and the total phospholipids were measured. The fatty acid compositions of cardiolipin and the total phospholipid fraction were determined. Comparative data from porcine heart and liver were obtained using the same methods. Differences in both the concentration and the fatty acid composition of the phospholipids were observed between the tissues. It appeared that the phospholipid bilayer was expanded relative to haem a in luteal mitochondria. It is proposed that in the ovary this expansion may be necessary to accommodate cytochrome P-450scc and its substrate, cholesterol.

Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome P-450 and cytochrome b5-dependent mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome P-450-dependent mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system.

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104-125 (1972) is characterized by high levels of glucose-6-phosphatase and 5'-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes b5 and P-450 as well as NADPH- and NADH-cytochrome c reductase activities are present but at lower levels than found in microsomes. Cytochrome c oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5'-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside triphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.

Isolated cytochrome c1 contains endogenous reducing equivalents. They can be removed by treating the protein with sodium dithionite followed by chromatography. This treatment has no effect on the reaction with cytochrome c, nor does it alter the optical spectrum, or the polypeptide or amino acid composition of the protein. Both the titration of dithionite-treated ferrocytochrome c1 with potassium ferricyanide and the anaerobic titration of dithionite-treated ferricytochrome c1 with NADH in the presence of phenazine methosulphate lead to the same value for the absorbance coefficient of cytochrome c1: 19.2 mM-1 . cm-1 at 552.4 nm for the reduced-minus-oxidised form. This value was also obtained when the haem content was determined by comparing the spectra of the reduced pyridine haemochromes of cytochrome c and cytochrome c1. Comparison of the optical spectra of cytochrome c and cytochrome c1 by integration shows equal transition moments for the transitions in the porphyrin systems of both proteins. A set of equations with which the concentration of the cytochromes aa3, b, c and c1 can be calculated from one reduced-minus-oxidised difference spectrum of a mixture of these proteins.

1. The fumarate reductase of Vibrio succinogenes, the terminal component of the electron transport chain of the bacterium, was extracted from the cytoplasmic membrane with Triton X=100 and purified to homogeneity (approx. 30-fold) by means of chromatograhy on hydroxyapatite and DEAE-Sephadex. The enzyme eluted from the ion-exchange column in two forms, one containing and the other lacking cytochrome b. 2. The enzyme lacking cytochrome b consisted of two peptides (Mr 79 000 and 31 000) which were present in a molar ratio of 1:1. The cytochrome-containing species contained an additional peptide (Mr 25 000) which was present in twice the molar amount of the others (2:1:1). 3. The hydrodynamic properties indicate that the functional enzymes consist of only one set of the corresponding peptides. 4. Each of the two enzyme molecules contains one protein-bound FAD which is linked to the Mr 79 000 peptide. Both enzyme species contain 9-10 mol iron-sulfur per mol of FAD which is associated with the Mr 79 000 and the Mr 31 000 peptide. Cytochrome b is present in an amount of 2 mol/mol of FAD, half of the cytochrome b has a midpoint potential of -20 mV. 5. The enzyme catalyzed two types of reaction. (a) Fumarate reduction by viologen radicals or anthrahydroquinonesulfonate, as well as succinate oxidation by ferricyanide or methylene blue, was independent of cytochrome b. (b) The activities of fumarate reduction by naphthohydroquinones and those of succinate oxidation by naphthoquinones were cytochrome b-dependent. This indicates that the electron transport from menaquinone to fumarate reductase in the membrane of the bacterium is mediated by a single component, cytochrome b (-20 mV). 6. The Km for fumarate was 0.35 mM with menadiol as the donor and that for succinate as 20 mM in the reverse reaction. Succinate oxidation was competitively inhibited by fumarate with a Ki of 0.35 mM.

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.

The complete amino acid sequence of rat cytochrome b5 has been determined. Isolation of this species of cytochrome b5 in its native form from microsomes by means of detergent solubilization required the inclusion of the protease inhibitor, phenylmethylsulfonyl fluoride, throughout the isolation steps. Omission of the protease inhibitor yielded a 97-residue heme-containing peptide without the membranous segment. The primary structure of the intact molecules was deduced from automated sequence analysis of peptides generated by proteolytic or chemical cleavage and isolated exclusively by reversed-phase HPLC. The blocked amino terminus of cytochrome b5 was identified as N-acetylalanine. The hexosamine content of the cytochrome preparation was less than 0.1 mol/mol protein, indicating an absence of asparaginyl linked oligosaccharide.

Isolated plasma membranes from the yeast Candida tropicalis grown on two different carbon sources (glucose or hexadecane), had similar contents of protein (60% of total dry weight), lipid (21-24%) and carbohydrates (16-21%). Sodium dodecyl sulphate gel electrophoresis of the membrane proteins revealed 17 and 19 protein bands, respectively, for glucose and hexadecane grown cells. There were marked differences in RF values and relative peak heights between the two gels. Sterols and free fatty acids were the major components of the plasma membrane lipids. Phospholipid content was less than 2% of total plasma membrane lipids. Membrane microviscosity, as determined by fluorescence polarization, was very high (16.6 P). Fatty acid determination of membrane lipids by gas chromatography showed a significant increase of C16 fatty acids in plasma membranes of cells grown on hexadecane. Reduced-oxidized difference spectra demonstrated the presence of a b-type cytochrome in both Saccharomyces cerevisiae and C. tropicalis plasma membranes. Its concentration in C. tropicalis plasma membranes was three-fold greater in cells grown on hexadecane than in glucose grown cells.

Methods for quantitation of catalase, cytochromes P-450 and b5 and mitochondrial cytochromes a + a3, b561 + b566, and c + c1 in isolated hepatocytes were developed in analogy to methods established for subcellular systems and were used to measure changes in specific hemoprotein concentrations due to pretreatment and to change in incubation conditions. Pretreatment of rats with phenobarbital or 3-methylcholanthrene resulted in increased concentrations of cytochromes P-450 and b5 on a cellular basis, but had no effect on the other hemoproteins. Chronic ethanol pretreatment resulted in increased cytochrome P-450 and decreased cytochromes a + a3 concentrations. Hemoprotein concentrations in hepatocytes decreased following 4-10-h incubations in rotating round-bottom flasks. Rates of decrease were dependent upon both incubation conditions and prior in vivo treatments with phenobarbital or 3-methylcholanthrene.

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of 'leaky' species; they are stable and can be maintained at 4 degrees C for many days without lysis. The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b5. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase. A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.

Of the six chlorophyll-protein complexes of spinach and barley resolved by mild gel electrophoresis, two were chlorophyll a-protein complexes of PS I, namely CP1a and CP1, which accounted for up to 30% of the total chlorophyll. Both of these complexes had one P-700 per 120 chlorophyll a molecules. Since spinach and barley thylakoids have some 400 chlorophyll molecules per P-700, these complexes may not have lost any of the chlorophyll associated with them in vivo. This may account for CP1a and CP1 having the characteristic low-temperature fluorescence normally associated with PS I in vivo, which is not found in complexes with low chlorophyll/P-700 ratios. Two-dimensional electrophoresis showed that all of the chlorophyll a and P-700 of CP1 was bound to 70 kilodalton polypeptides. The PS I reaction centre complex of lowest mobility, CP1a, contained CP1 and four additional low molecular weight polypeptides. The three light-harvesting complexes resolved had major 25 and 23 kilodalton polypeptides. The presumed reaction centre complex of PS II contained major 50 and 47 kilodalton polypeptides.

A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

Mitochondria isolated from rats chronically fed ethanol demonstrated a marked inability to produce energy. The respiratory control ratio, the ADP/O ratio and state 3 respiration rates were all decreased. Coupled with other data, a progression of ethanol-induced changes is proposed with site I being altered prior to site II. Quantitation of mitochondrial cytochromes revealed decreases in cytochromes b and aa3 and an increase in c1. Evaluation of respiration activity in relation to temperature showed ethanol-induced changes in the transition temperature (Tf) which may have been related to changes in the lipid composition of the inner membrane. Mitochondrial membranes were separated, and analysis of fatty acids and phospholipids was performed. Various fatty acids were altered in both membranes; however, the outer membrane was altered more severely. A decrease in the arachidonate : linoleate ratio was observed only in the outer membrane; however, there was no ethanol-induced change in degree of unsaturation in either membrane. Phospholipid quantitation showed a reduction of total lipid phosphorous/mg protein in both membrane fractions; however, the inner membrane was most affected. Cardiolipin was the only phospholipid in this membrane which remained unaltered. The evidence indicates that the mechanism for ethanol-induced damage to the liver mitochondrion involves lipid compositional changes as well as changes in cytochromes and possibly other proteins.

The membrane-bound cytochrome f-556.5 from the blue-green alga Spirulina platensis was purified to apparent homogeneity. Most of its properties are comparable to cytochrome f isolated from higher plants and green algae. It is clearly distinguishable from soluble cytochrome c-554, also present in Spirulina, which probably replaces the function of plastocyanin in photosynthetic electron transport. 1. The reduced form of cytochrome f exhibits an asymmetrical alpha-band with a maximum at 556.5 nm, and a pronounced shoulder at 550 nm. The beta-, gamma and delta-bands coincide with those described for Scenedesmus cytochrome f-553, with maxima at 524 (532), 422, 331 and a protein peak at 276 nm. The maximum of ferricytochrome f is at 410.5 nm; there is no indication of a weak 695 nm band, described for soluble c-type cytochromes. The purest preparations had a delta/protein-peak ratio of 0.8; the gamma/alpha ratio was 7.3. Formation of a pyridine hemochromogen with a maximum at 550 nm indicated a c-type cytochrome. The molar extinction coefficient at 556.5 nm is 30200, the differential extinction coefficient 21 500. 2. The molecular weight determined by gel filtration or SDS-polyacrylamide gel electrophoresis is 33 000 and 34 000, respectively. 3. The redox properties differ from those described for other cytochromes f isolated from green algae and higher plants: the midpoint redox potential is significantly more negative (+318 mV, pH 7.0) and from pH 6 to 10 no pH dependence is observed. 4. The isoelectric point was determined at pH 3.95, which is more acidic as compared to other cytochromes f. 5. Comparison of the amino acid composition indicated a distant relationship to higher plant cytochrome f and a closer relationship to cytochrome f from green algae.

The "Triton Subchloroplast Fraction I" or "TSF-I particles" can be further fractionated into a cytochrome fraction and a P-700-containing fraction essentially free of cytochromes. The cytochrome complex contains cytochromes f and b6 in approx. equimolar amounts, and, in addition, also plastocyanin and one iron-sulfur protein, all in the bound state. Bound plastocyanin was characterized by EPR spectroscopy. The EPR spectrum of the bound iron-sulfur protein resembles that previously detected in Phostosystem I particles under highly reducing conditions at lower than -560 mV. The redox potential of P-700 in the cytochrome-free high-P-700 particles was measured to be +468 mV; those of cytochromes f and b6 are +345 and -140 mV, respectively. Among the four components present in the complex, only cytochrome f can be coupled to a Photosystem I particle and undergoes photooxidation. This coupled photooxidation is totoally inhibited by KCN and only partially inhibited by HgCl2. The similarity of the complex containing cytochromes f and b6, plastocyanin, and an iron-sulfur protein to complexes III and IV of the mitochondrial respiratory redox chain and a possible involvement of the complex in cyclic photophosphorylation are noted and discussed.

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.

We have recently reported that with a linear sucrose density gradient centrifugation two distinct types of membrane fragments, designated as X- and Y-fragments are obtained (Huang, C. H., Keyhani, E. and Lee, C. P. (1973) Biochim. Biophys. Acta 305, 455-473). Further characterization of these two membranes fragments is reported. (1) Potassium chloride at the concentration of 0.15 m extracts 7% and 30% of cytochrome c from the X- and Y-fragments, respectively. (2) When cytochrome c was added to the mitochondrial suspension prior to sonication, the cytochrome c content was increased by 6-8-fold in both X- and Y-fragments. Subsequently KC1 extraction resulted in loss of cytochrome c by 1/4 in the X- and by 2/3 in the Y-fragments. (3) With partially inhibitory concentrations of KCN, cytochrome c in either the X- or the KC1 extracted X-fragments showed uncoupler-sensitive, biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. Under identical conditions rapid first order reduction kinetics were seen for cytochrome c in Y-fragments supplemented with either oligomycin or oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (4) When cytochrome c was added to the mitochondrial suspension after sonication, a significant amount of cytochrome c was bound to both X- and Y-fragments, but was readily removed with a high ionic strength medium. (5) Lubrol had little effect on the ATPase activity of the X- and the Y-fragments, suggesting a lack of membrane-buried ATPase. (6) Partial depletion of ATPase in X-fragments did not induce an increase in reactivity towards externally added cytochrome c. (7) Both the X- and the Y-fragments showed an energy-linked fluorescence enhancement of 8-anilinonaphthalene-1-sulfonate and an energy-linked fluorescence decrease of quinacrine. (8) In the presence of K-+ nigericin alone or in combination with valinomycin exhibited a stimulating effect on the rate of NADH oxidase of the oligomycinsupplemented X- and Y-fragments.

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.

Mouse myeloma cells and mitochondria had the same kinds of cytochrome components in the respiratory chain as the normal ones. Their constitution, however, was abnormally different from that found in normal cells and mitochondria. The cytochrome aa3 concentration was especially low in the myeloma as compared with cytochrome c concentration, and the resulting cytochrome aa3/c ratio was 0.25, which was the lowest ever reported in animal mitochondria. Normal lymph node cells, producing the immunoglobulin similar to the myeloma cells, had a ratio of 1.1. Human myeloma mitochondria had the same characteristics as the mouse myeloma. Ascite form myeloma originated from mouse solid from myeloma grew faster, and yet aa3/c of 0.5 in the ascites myeloma was found to be quite similar to that observed in various ascites tumor cells such as hepatomas, Ehrlich and sarcoma 180. A significant part of the cytochromes in the respiratory chain of the mouse myeloma remained in the oxidized form in the cyanide-inhibited or anaerobic states, and was reduced only by the addition of dithionite. The properties of the b cytochromes in mouse myeloma mitochondria are also described and discussed in the context of multiple forms of the b cytochromes in the respiratory chain.

In order to evalute the effectiveness and selectivity of various reagents in the solubilization of bacterial membranes, membranes of Micrococcus lysodeikticus were treated with detergents and chaotropic agents. The composition of the extracts so obtained was analyzed by rocket and two-dimensional immunoelectrophoretic techniques. Recoveery of succinate-, malate-, and reduced nicotinamide adenine dinucleotide- (NADH) dehydrogenases, ATPase, succinylated lipomannan and cytochromes in the extracts was measured. Treatment with a variety of non-denaturing detergents produced extracts that were generally qualitatively uniform although quantitative differences were observed. The degree of extraction of various components was correlated with the hydrophile-lipophile balance. Several chaotropic agents were also evaluated as reagents for membrane solubilization. These agents were less effective in extraction of bulk protein, but produced extracts enriched in some membrane components.

Particulate fractions of Escherichia coli have been submitted to electrophoretic fractionation in a buffer stabilized by sucrose gradient. Inner membrane and outer membrane were readily resolved. A combination of electrophoresis, fractional centrifugation and gel filtration can remove remaining contamination by ribosomes and cytoplasm. The presence of particles containing no phospholipids was detected after differential centrifugation. The nature of this fraction is unknown. The inner membrane exhibited heterogeneity on electrophoresis.

1. Three nuclear mutants of Saccharomyces cerevisiae deficient in succinate dehydrogenase have been isolated. Two of these mutants are allelic. 2. The amount of covalently bound flavin of submitochondrial particles of the two allelic mutants is about 14% and that of the third mutant about 50% of the amount in wild-type particles. The turnover number of succinate dehydrogenase of particles is decreased in all mutants. The turnover number of fumarate reductase is increased in the two allelic mutants, but decreased in the third mutant. 3. EPR spectra, measured at 82 degrees K, show that the amplitude of the g equals 1.93 signal in particles of the two allelic mutants is less than 10% of that in wild-type particles. It is concluded that iron-sulphur centres other than those of succinate dehydrogenase make only a negligible contribution to the line at g equals 1.93 in wild-type particles. 4. EPR measurements below 20 degrees K show that the amplitude of the signal at g equals 2.01 detected in oxidized particles is decreased in particles of the two allelic mutants. 5. A signal with lines at g equals 2.027 and g equals 1.933 is detected at low temperatures in all particle preparations, even in those from a cytoplasmic petite mutant. It is suggested that this signal is derived from a contaminant and not from the inner membrane.

Cardiolipin and cytochrome aa3 contents of isolated plant cells (sycamore cells) and their purified mitochondria were measured. Since the cardiolipin/cytochrome aa3 ratio was the same in the intact cells and in the isolated mitochondria it was strongly suggested that cardiolipin is present only in the mitochondria. Furthermore, outer and inner mitochondria membranes of purified sycamore cells and mung bean hypocotyl mitochondria were separated and it was shown that cardiolipin is localized in the inner mitochondrial membrane.

The resonance Raman spectrum of turnip cytochrome f is similar to that of other c-type cytochromes with the exception of a single band at 1532 cm-1 which is shifted to lower frequency relative to its position (1542-1545 cm-1) in other c-type cytochromes. Comparison of the frequency of this band with that in alkylated cytochrome c at high pH suggests that the sixth heme iron ligand in cytochrome f is a deprotonated lysine amino group rather than a methionine sulfur. Comparison of the amino-acid sequences of cytochromes f and c1 suggests lysine-145 as a likely candidate for the sixth heme iron ligand in cytochrome f.

A ubiA- menA- double quinone mutant of Escherichia coli K12 was constructed together with other isogenic strains lacking either ubiquinone or menaquinone. These strains were used to study the role of quinones in electron transport to oxygen and nitrate. Each of the four oxidases examined (NADH, D-lactate, alpha-glycerophosphate and succinate) required a quinone for activity. Ubiquinone was active in each oxidase system while menaquinone gave full activity in alpha-glycerophosphate oxidase, partial activity in D-lactate oxidase but was inactive in NADH and succinate oxidation. The aerobic growth rates, growth yields and products of glucose metabolism of the quinone-deficient strains were also examined. The growth rate and growth yield of the ubi+menA- strain was the same as the wild-type strain, whereas the ubiA-men+ strain grew more slowly on glucose, had a lower growth yield (30% of wild type) and accumulated relatively large quantities of acetate and lactate. The growth of the ubiA-menA- strain was even more severely affected than that of the ubiA-men+ strain. Electron transport from formate, D-lactate, alpha-glycerophosphate and NADH to nitrate was also highly dependent on the presence of a quinone. Either ubiquinone or menaquinone was active in electron transport from formate and the activity of the quinones in electron transport from the other substrates was the same as for the oxidase systems. In contrast, quinones were not obligatory carriers in the anaerobic formate hydrogenlyase system. It is concluded that the quinones serve to link the various dehydrogenases with the terminal electron transport systems to oxygen and nitrate and that the dehydrogenases possess a degree of selectivity with respect to the quinone acceptors.

The polypeptide composition of thylakoid membrane fractions from the heterotrophic cyanobacterium Aphanocapsa 6714 was examined by electrophoretic and immunoblotting procedures. We have identified thylakoid cytochromes f, b6, c-550 and c-553 by tetramethylbenzidine staining of lithium dodecyl sulfate polyacrylamide gels; we also have identified the Rieske Fe-S center protein and subunit 4 of the cytochrome b6/f complex. We have characterized phycobilisomes and active core preparations of PS I and PS II. PS I is comprised of five polypeptides (62 kDa, 14.5 kDa, 10 kDa, and two proteins of less than 10 kDa), and our PS II preparation is highly enriched for three chlorophyll-binding proteins of 48, 45 and 36 kDa. Furthermore, we have resolved the chlorophyll-binding complexes on non-denaturing gels and have determined the polypeptide composition of each chlorophyll-containing band. Three bands are associated with PS I (I, IIa and IIb) and three bands are PS II components (III', IIIa and IIIb) as judged by low-temperature fluorescence emission spectra. Band III' contains a 64 kDa antenna polypeptide, IIIa contains the 48 kDa and 45 kDa polypeptides, and IIIb is comprised solely of a 36 kDa protein. The IIIb apoprotein represents a novel PS II component; its possible role in photochemistry is discussed.

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.

The spectral properties of sympathetic nerve vesicles isolated from the vas deferens of the rat are similar to those of the bovine chromaffin granule membranes and bovine nerve trunk vesicles, indicating the presence of the specific cytochrome b-561. The cytochrome occurs only in the fractions containing nerve vesicles, thus suggesting usefulness as a marker enzyme.