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l -(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l -glutamate oxidation is not dependent on externally added NAD + , NADP + , FAD, or FMN for activity. O 2 , phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l -glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l -(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l -amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l -glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2- n -heptyl-4-hydroxyquinoline- N -oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R 3 fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome ( a 2 , a 1 , b 1 , c 4 and c 5 ) are reduced by the addition of l -glutamate.

The amino acid sequence of the cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015 (Iwasaki's ‘Pseudomonas denitrificans’) has been determined. This organism is the only non-photosynthetic bacterium in which the protein has been found. The protein consists of a single polypeptide chain of 127 residues, with a single haem covalently attached to two cysteines. Unlike normal cytochromes c, the haem attachment site is very close to the C-terminus. The amino acid sequence around the haem attachment site is very similar to that of Chromatium vinosum D cytochrome c′. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50022 at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

1. 1. The amino acid sequence of Struthio camelus cytochrome c was derived by sequencing tryptic peptides. 2. 2. The sequence differs from that of the typical bird cytochrome c in a single position. 3. 3. Phylogenetic studies were carried out to compare the positioning of chicken, turkey, Pekin duck, penguin, pigeon and ostrich according to their cytochrome c structures.

The branched-chain sugar d-apiose was oxidized to CO2 by both Lemma minor and a bacterium which was isolated from the surface of germinating parsley seeds. An inducible dehydrogenase which catalyzed the interconvension of d-apiose and d-apiitol was detected in extracts of this microorganism. The enzyme which was purified about 200-fold was specific for d-apiose and d-apiitol. It oxidized myo-inositol and meso-erythritol slowly, but it was completely inactive with all of the other sugars and polyols tested. The enzyme was specific for NAD+ and NADH as electron acceptor and donor, respectively. NADP+, NADPH, ascorbate, FAD, FADH2, cytochrome c and ferricyanide were inactive. The Km for D-apiitol was 1.16·10-2 M, d-apiose was 7.14 · 10−2 M, NAD+ was 3.5 · 10-4 M and NADH was 1.5–15-5 M. At high concentrations NADH inhibited the reaction. The molecular weight of the dehydrogenase determined by chromatography on Sephadex 200 and sucrose density centrifugal was 110 000. The products of the reaction were characterized by paper chromatography, periodate oxidation and gas chromatography of acetylated derivatives. A colorimetric method for the quantitative determination of small amounts of d-apiose was also developed during the course of this study.

Proteolysis of horse heart cytochrome C with pepsin for 3 min produces a large heme peptide, which was shown to be a mixture of the sequences 1–64 and 1–66. By ion exchange chromatography, gel filtration and preparative paper chromatography the heme-free fraction was resolved into seven peptide fragments, corresponding to sequences 65–94, 65–96, 67–94, 67–96, 67–80, 95–104 and 97–104.

Twenty-seven peptides, including the haemopeptide, were isolated from a single chymotryptic digest of seal heart cytochrome c by chromatography on Sephadex G-75, Whatman phosphocellulose P-70 and Bio-Rad AG 50W-X2. These peptides were purified by electrophoresis or chromatography on paper and their sequences were determined. The complete sequence of the protein was deduced by alignment of these peptides by comparison with the known sequences of several other cytochromes c. Seal cytochrome c differs from the dog and horse proteins in 1 and 6 positions, respectively. The single difference from dog cytochrome c is found in position 100 where isoleucine, in the seal, replaces the lysine found in the dog.

Proposed amino acid sequences of cytochrome c from nasturtium (Tropaeolum majus L.), box-elder (Acer negundo L.), elder (Sambucus nigra L.) and parsnip (Pastinaca sativa L.) are presented. Because of the very limited amounts of cytochrome available from some plant sources, peptides derived from the cytochromes c have been sequenced by the semi-quantitative dansyl–Edman technique (Gray & Hartley, 1963) without supporting quantitative amino acid analyses. Because of the qualitative nature of the work, the sequences proposed must be regarded as tentative. Considerations of homology, although useful as a guide, have been kept to a minimum in the construction of sequences. Only the nasturtium sequence relies on considerations of homology for a complete ordering of the peptides. Where material permitted, each residue of a proposed sequence was determined at least once from both a tryptic and a chymotryptic peptide.

Cytochrome c 553 of Desulfovibrio , vulgaris differs in amino acid composition and in molecular weight (9, 100 ± 100) from cytochrome c 3 (12,000) of the same organism. This protein can be easily obtained in crystalline form. It consists of a single polypeptide chain comprising about 80 amino acid residues and bearing a single heme group. Both termini are freely available; alanine and leucine are marking the amino terminal and the carboxyl terminal position, respectively. In addition to cytochrome c 553 and to cytochrome c 3 a third c-type cytochrome, of larger molecular weight, was detected in D. , vulgaris and related organisms.

An antibody to Clostridium pasteurianum rubredoxin was found in goat serum after multiple injections of the protein. This antibody was purified by adsorption and elution from a Sepharose-rubredoxin column. The purified antibody formed a precipitating complex with C. pasteurianum rubredoxin and aporubredoxin, but not with the rubredoxin from Micrococcus aerogenes, Peptostreptococcus elsdenii , and Pseudomonas oleovorans . All rubredoxins tested were adsorbed to Sepharose-antirubredoxin columns indicating that each could form a soluble complex with anti- C. pasteurianum rubredoxin. The relative affinity of the antirubredoxin for the various rubredoxins was demonstrated by its ability to inhibit the rubredoxin-dependent reduction of cytochrome c by NADPH in the presence of NADP-ferredoxin oxidoreductase. These data suggest that there are two antigenic determinants in C. pasteurianum rubredoxin and only one such determinant in the rubredoxin from other organisms which are recognized by anti- C. pasteurianum rubredoxin.

A soluble cytochrome was isolated and purified from the slime mould Physarum polycephalum and identified as cytochrome c by room-temperature and low-temperature (77 degrees K) difference spectroscopy. A close similarity between P. polycephalum and mammalian cytochromes c was suggested by a comparison of the initial rates of oxidation of both proteins by mammalian mitochondria. This similarity was further emphasized by redox titrations and gel-electrophoretic studies which indicated that P. polycephalum cytochrome c has an oxidation-reduction midpoint potential of +257mV at pH7.0 and a molecular weight of 12500+/-1500 (mean+/-maximum deviation for a set of six measurements). P. polycephalum exhibits an absolute requirement for protohaemin for growth. The (59)Fe-labelled haemin was prepared by chemical synthesis from protoporphyrin. The purified product had a specific radioactivity of 0.8+/-0.02muCi/mol. Growth of P. polycephalum in the presence of [(59)Fe]haemin resulted in the incorporation of (59)Fe into the plasmodial cytochrome c. The specific radioactivity of the cytochrome c haem was 0.36+/-0.02muCi/mol. The high specific radioactivity of the cytochrome haem indicates that synthesis of the holoenzyme must proceed by direct attachment of haem to the apoprotein rather than by the intermediate formation of a protoporphyrinogen-apoprotein complex. The observed decrease in the specific radioactivity of the haem group is attributed to exchange of the (59)Fe with unlabelled iron in the plasmodia either before or during attachment of the haem group to the apoprotein.

The amino acid sequence of tomato (Lycopersicon esculentum Mill.) cytochrome c was determined using 2 μmoles of protein. Analysis of tryptic and chymotryptic peptides by Gray's method (8) showed that the protein consists of a single chain of 111 residues and is homologous with other previously determined plant mitochondrial cytochromes c.

Two heterotrophic bacteria that oxidized thiosulfate to tetrathionate were isolated from soil. The enzyme system in one of the isolates (C-3) was constitutive, but in the other isolate (A-50) it was induced by thiosulfate or tetrathionate. The apparent K m for oxygen for thiosulfate oxidation by A-50 was about 223 μ m , but, for lactate oxidation by A-50 or thiosulfate oxidation by C-3, the apparent K m for oxygen was below 2 m m . The oxidation of thiosulfate by A-50 was first order with respect to oxygen from 230 μ m . The rate of oxidation was greatest at p H 6.3 to 6.8 and at about 10 m m thiosulfate, and it was strongly inhibited by several metal-binding reagents. Extracts of induced A-50 reduced ferricyanide, endogenous cytochrome c , and mammalian cytochrome c in the presence of thiosulfate. A-50, once induced to oxidize thiosulfate, also reduced tetrathionate to thiosulfate in the presence of an electron donor such as lactate. The optimal p H for this reaction was at 8.5 to 9.5, and the reaction was first order with respect to tetrathionate. There was no correlation between the formation of the thiosulfate-oxidizing enzyme of A-50 and the incorporation of thiosulfate-sulfur into cell sulfur. Thiosulfate did not affect the growth rate or yield of A-50.

1. The oxidation of NADH and NADPH catalysed by the soluble supernatant from the hepatopancreas of Octopus vulgaris is due to a single enzyme, which has been purified approximately 100-fold. The enzyme reacts rapidly with potassium ferricyanide, and more slowly with 2,6-dichlorophenol-indophenol. No activity is obtained with oxygen, cytochrome c, lipoic acid, vitamin K1, vitamin K3, ubiquinone-30, p-benzoquinone, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride or methylene blue. 2. GSH, cysteine and mercaptoethanol stimulate the enzymic activity up to fivefold. GSSG is without any apparent effect. When stimulated by GSH the enzyme becomes sensitive to dicoumarol, which produces an inhibition competitive with respect to the activator. 3. The purified enzyme contains an acid-removable flavine component, which has been identified as FMN by spectrofluorimetry and chromatography in three solvent systems. After acid ammonium sulphate treatment the enzymic activity is lost, but it can be almost fully restored by incubation with FMN. FAD produces only a partial reactivation.

A simple and rapid method is described for the isolation of the desmosines from elastin. Elastin is hydrolyzed with 6 N HCl in a conventional manner, treated with decolorizing charcoal, and adjusted to pH 2.8. Gel permeation on Bio-Gel P-2 resolves an ultraviolet-absorbing peak eluted between cytochrome c and cyanocobalamin. The desmosines are recovered by evaporation of the appropriate fractions. The method is suitable for quantities ranging from 20 mg to 5 gm elastin.

The amino acid sequence of cytochrome c from Nigella damascena L. was determined on 0.2μmol of protein. Peptides from a single chymotryptic digest were analysed by the dansyl–Edman procedure. These peptides were ordered by reference to the sequences of other plant cytochromes c, assuming that the Nigella cytochrome is homologous with the other cytochromes. Many of the Nigella peptides were one or two residues short when compared with the corresponding chymotryptic peptides from other plant cytochromes c. These residues are assumed to have been removed by an endogenous carboxypeptidase, and the identification and placing of these residues is entirely based on homology. These residues are numbered 3, 18, 42, 43, 44, 54, 67, 72, 73, 82 and 105. A number of other positions are almost entirely placed by homology. These are positions which could not be placed definitely by dansyl–Edman analysis or by dansylation after digestion with carboxypeptidase A, and are numbered 14, 15, 16, 39, 40, 85, 86, 87 and 88. Except for residue 15, all residues based entirely, or nearly so, on homology have been previously found invariant in sequences of plant cytochromes c. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50017, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

1. The effects of cyanogen bromide on horse-heart cytochrome c and horse-heart myoglobin have been investigated. Cytochrome c yielded four fragments, of which two were haemopeptides. The two colourless peptides had amino acid compositions corresponding to those that are expected, on the basis of the sequence proposed for horse-heart cytochrome c by Margoliash, Smith, Kreil & Tuppy (1961), from cleavage at both methionine residues. Of the two haemopeptides, one was isolated and shown to be that derived from cleavage at only one methionine residue, that nearer to the C-terminus of the peptide chain. 2. Myoglobin also gave four peptides, three of which accounted for the total amino acid content of the intact protein. The fourth fragment arose by cleavage at a single methionine residue, that nearer the C-terminus. Characterization of this fourth fragment made it possible to deduce the order of arrangement of the fragments in the intact molecule.

Cobalt cytochrome c has been prepared from porphyrin cytochrome c in water/acetic acid solvent. The dominant band in the electrophoresis of the product at pH7 has the same mobility as the native protein. Dithionite changes the ultraviolet/visible spectrum markedly and generates an epr signal with cobalt hyperfine and other superhyperfine features. Nitric oxide removes part of the epr signal. Fractionation on Amberlite CG-50 under NaCl gradient at pH8.0 yields two major components distinguished by their rates of reactivity to dithionite and electrophoretic mobility. Cobalt cytochrome c is reduced by DPNH cytochrome c reductase to produce the same electron paramagnetic resonance signal as that generated by dithionite.

The nonsulfur purple bacterium Rps. palustris was adapted to grow photoautotrophically with thiosulfate as substrate. An isolated cell-free fraction catalyzed the enzymatic transfer of electrons from thiosulfate to endogenous and/or added mammalian cytochrome c . Antimycin A, NOQNO, rotenone, amytal and atebrin did not inhibit the thiosulfate-cytochrome c reductase. The products of thiosulfate oxidation were primarily tetrathionate, trithionate, and sulfate, suggesting oxidation via the polythionate pathway. Succinate, formate and NADH were also effective electron donors in this system showing Michaelis constants of 40, 30 and 0.025 m m , respectively for cytochrome c reduction. The NADH-cytochrome c reductase was not inhibited by flavoprotein inhibitors and by Antimycin A or NOQNO. The cell-free extracts also contained an active cytochrome c -O 2 oxidoreductase which was inhibited by cyanide, azide and EDTA, and these inhibitions were overcome by the addition of Cu 2+ . The oxidase activity was stimulated by the addition of uncoupling agents such as CCCP and DNP, as well as by Antimycin A and NOQNO. Reduced + CO minus reduced difference absorption spectra revealed the presence of cytochrome components of the a and o types which may function as the terminal oxidase(s).

1. 1. The concentration of cytochrome a + a 3 and other cytochromes and their prosthetic groups have been measured in resting anaerobically grown Sacchromyces cerevisiae “yeast foam” before and after adaptation to O 2 . The increase in concentration of haem a and other haems parallels the increase in the haemoproteins in the adapted yeast. 2. 2. The incorporation of [2- 14 C]glycine and δ-amino[4- 14 C]laevulinic acid into haem a and other cytochrome prosthetic groups confirms that there is substantial biosynthesis de novo of haem a , cryptohaem a and other haems in adapting yeast. The labelled glycine is equally available for incorporation into the haem and protein moieties of cytochrome c . 3. 3. The uptake of [2- 14 C]glycine into the withdrawal from the amino acid pool during adaptation has been measured. The concentration of glycine and other amino acids, excepting glutamic acid, is considerably diminished during adaptation. Glutamic acid had accumulated by the end of adaptation. 4. 4. After development of the cytochromes porphyrin synthesis continued, the main porphyrin being coproporphyrin. A new tetrapyrrole, coprobiliverdin, the formation of which is likely to be different from that of biliverdin, is also excreted from the yeast cells.

In an in vitro system consisting of rat liver microsomes and NADPH, significant lipid peroxidation was observed along with a concomitant loss of cytochrome P-450. This spectral loss of cytochrome P-450 was shown to be the result of a breakdown of cytochrome P-450 heme. Inhibitors of lipid peroxidation also prevented the loss of cytochrome P-450, demonstrating a direct relationship between lipid peroxidation and breakdown of cytochrome P-450 heme. This breakdown of cytochrome P-450 heme during lipid peroxidation is unrelated to the degradation of cytochrome P-450 heme by an active metabolite of certain allyl-containing barbiturates and barbiturate related compounds. In addition, it appears that different breakdown products of heme are produced by these two mechanisms.

1. 1. N-Methyl-N′-nitro-N-nitrosoguanidine labeled with 14C at the guanidino-carbon was incubated with horse heart cytochrome c in 20 mM cacodylate buffer (pH 7.0). Preparations of modified cytochrome c associated with 1.0, 2.0, 3.4, 6.9, 8.0, 13.5 and 14.5 moles of nitroamidino groups per mole were separated by Amberlite CG-50 and DEAE-cellulose column chromatographies. 2. 2. The spectrophotometric characters in the visible region of modified cytochrome c with 3,4, 8.0 or 13.5 moles of nitroamidino groups were identical to those of native cytochrome c in the reduced and oxidized forms. 3. 3. Modified cytochrome c was more negatively charged than native cytochrome c as shown by electrophoresis on a cellulose acetate membrane. 4. 4. The oxidation-reduction potential of modified cytochrome c with 8.0 moles or nitroamidino groups was 270 mV while that of native cytochrome c was 255 mV at pH 6.5. 5. 5.Modified cytochrome c reduced by dithionite. However, its rate of enzymatic reduction by NADH with rat liver mitochondria was less than that of native cytochrome c. 6. 6. Cytochrome c modified with [guanidino-14C]MNNG was hydrolyzed enzymatically and the radioactive compound isolated was identified as nitrohomoarginine.

A flavin obtained from Chromatium cytochrome c -552 has been purified and characterized with respect to its neutral and acid absorption spectra, fluorescence-pH relationship, and behavior on partition and molecular exclusion chromatography. The chromatographic results are consistent with a flavin adenine dinucleotide type structure. Neutral and acid absorption spectra taken after degradation of the flavin to the riboflavin level differ in the near UV from those of riboflavin and are similar to those of flavins modified in the dimethylisoalloxazine ring system, such as the histidyl-riboflavin obtained from mammalian succinate dehydrogenase. However, cytochrome c -552 flavin when at the riboflavin level and histidyl-riboflavin are quite dissimilar when compared fluorometrically and by molecular exclusion chromatography.

IT is now known that the phagocytic process in polymorphonuclear leucocytes (PMN) is accompanied by marked increases in the rates of oxygen consumption, glucose oxidation by the hexosemonophosphate shunt (HMPS) and oxidation of formate to carbon dioxide1–3. The energy for the phagocytic process seems to come from glycolysis1. The rate of glucose oxidation by the shunt in leucocytes seems to be regulated by the intracellular concentration of NADP (refs. 3 and 4). A suitable hypothesis for the enzyme basis underlying the metabolic stimulation in PMN during phagocytosis should therefore include a mechanism for the reoxidation of NADPH. Attempts have been made to correlate the stimulated activity of the hexosemonophosphate shunt in phagocytosing leucocytes with increased oxidation of NADPH. The evidence indicates that oxidation of NADPH in phagocytosing leucocytes is not accomplished by the classical route of hydrogen transport by way of cytochrome reductase and cytochrome c (ref. 1). Evans and Karnovsky5 have presented evidence for the existence in PMN cell extracts of an NADP-linked lactate dehydrogenase which is activated at low pH. Results obtained by Iyer, Islam and Quastel2 indicated that PMN possess an enzyme system capable of oxidizing NADPH and NADH by a reaction involving the formation of hydrogen peroxide. The enzyme, however, was found to bo much more active towards NADPH than NADH and its activity was strongly enhanced by manganese ions. Later Roberts and Quastel6 reported that the NADPH oxidase in PMN was probably peroxidase. Based on the available evidence, a mechanism was proposed to account for the metabolic changes observed in PMN during phagocytosis. This is seen as follows.

Of the hæmatin compounds present in the cell, component c of cytochrome alone can be readily obtained in solution by extraction with water of either dry or acetone yeast (Keilin, 1925). Recently a method for extracting and concentrating this pigment from yeast has been described, and it was found that the component c thus obtained has the same properties as it has in intact living cells or in the extracts of dry of acetone yeast cells (Keilin, 1930, pp. 421-423). It was shown previously that cytochrome c is a hæmochromogen which differs from the usual hæmochromogen compounds in two important properties, namely, in not being autoxidisable and in not forming a carbon monoxide compound. It was also shown that, without changing it absorption spectrum, the component c can be easily modified in such a way as to show the properties of an ordinary hæmochromogen (Keilin, 1925, 1926). As the various hæmochromogens obtained from cytochrome c have the positions of the absorption bands very different from those of protohæmochromogens it was clear that the iron-porphyrin portion of its molecule is completely different from the ordinary protohæmatin. A series of facts discussed in previous papers suggested, however, that cytochrome originated from the ordinary protohæmatin which is also present in all cells of aerobic organisms. The object of the experiments described in this paper is the study of the porphyrin portion of cytochrome c and its relationship to other prophyrins, especially to protoporphyrin.