Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.     

Cytochrome c1 from Paracoccus denitrificans

Cytochrome c1 was purified from the bacterium Paracoccus denitrificans. It is an acidic, hydrophobic polypeptide with an apparent molecular weight of around 65000 and a single, covalently attached heme; it cross-reacts immunologically with cytochrome c1 from yeast mitochondria. The amino acid sequence of the tryptic heme peptide of the bacterial cytochrome c1 shows extensive homology to the corresponding region of beef heart cytochrome c1 [Wakabayashi, S. et al. (1982) J. Biol. Chem. 257, 9335-9344]. Positive evidence for a stable association of the Paracoccus cytochrome c1 with other polypeptides and b-type heme components ('bc1-complex') has not yet been obtained.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.     

Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans

Highly active succinate-ubiquinone reductase has been purified from cytoplasmic membranes of aerobically grown Paracoccus denitrificans. The purified enzyme has a specific activity of 100 units per mg protein, and a turnover number of 305 s-1. Succinate-ubiquinone reductase activity of the purified enzyme is inhibited by 3'-methylcarboxin and thenoyltrifluoroacetone. Four subunits, with apparent molecular masses of 64.9, 28.9, 13.4 and 12.5 kDa, were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains 5.62 nmol covalently bound flavin and 3.79 nmol cytochrome b per mg protein. The 64.9 kDa subunit was shown to be a flavoprotein by its fluorescence. Polyclonal antibodies raised against this protein cross-reacted with the flavoprotein subunit of bovine heart mitochondrial succinate-ubiquinone reductase. The 28.9 kDa subunit is likely analogous to the bovine heart iron protein, and the cytochrome b heme is probably associated with one or both of the low-molecular-weight polypeptides. The cytochrome b is not reducible with succinate but is reoxidized with fumarate after prereduction with dithionite. Iron-sulfur clusters S-1 and S-3 of the Paracoccus oxidoreductase exhibit EPR spectra very similar to their mitochondrial counterparts. Paracoccus succinate-ubiquinone reductase complex is thus similar to the bovine heart mitochondrial enzyme with respect to prosthetic groups, enzymatic activity, inhibitor sensitivities, and polypeptide subunit composition.     

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.     

An atypical heme-binding structure of cytochrome c1 of Euglena gracilis mitochondrial complex III

Complex III was purified from submitochondrial particles prepared from Euglena gracilis. The purified complex consisted of 10 subunits and lost antimycin sensitivity. The Euglena complex III showed an atypical difference absorption spectrum for cytochrome c1 with its alpha-band maximum at 561 nm. The pyridine ferrohemochrome prepared from covalently bound heme in the Euglena complex III had an alpha-peak at 553 nm. This wavelength is the same as that of pyridine ferrohemochrome prepared from Euglena mitochondrial cytochrome c (c-558), the heme of which is linked to only a single cysteine residue through a thioether bond. Cytochrome c1 which was a heme-stained subunit with a molecular mass of 32.5 kDa was isolated from the purified complex III and its N-terminal sequence of 46 amino acids was determined. On the basis of apparent homologies to cytochromes c1 from other sources, this sequence included the heme-binding region. However, the amino acid at position 36, corresponding to the first cysteine involved in heme linkage in other cytochromes c1, was phenylalanine. Position 39, corresponding to the second cysteine, was not identified despite the treatment for removal of the heme and carboxymethylation of the expected cysteine. The unidentified amino acid is assumed to be a derivative of cysteine to which the heme is linked through a single thioether bond. The histidine-40 corresponding to the probable fifth ligand for heme iron was conserved in Euglena cytochrome c1.     

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus)

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.     

Isolation and partial charcterization of a cytochrome b complex and cytochrome oxidase from yeast mitochondria.

A cytochrome b complex and cytochrome oxidase have been purified 14- and 20-fold respectively from yeast submitochondrial particles by a simple procedure involving their spontaneous precipitation from a deoxycholate extract. The recovery of both proteins was almost quantitative. The specific heme contents were 11 and 8 nmoles/mg protein for the cytochrome b complex and cytochrome oxidase respectively and both were spectrally pure. Sodium dodecyl sulfate gel electrophoresis resolved the cytochrome b complex into seven distinct subunits with molecular weights 42,000, 33,000, 27,500, 23,000, 15,500, 13,000 and 10,500. Cytochrome oxidase contained five bands with molecular weights 42,000, 26,500, 21,000, 14,000 and 10,500.     

Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

Isolation and characterization of cytochrome C1 from photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Cytochrome c1 of photosynthetic bacterium R. sphaeroides R-26 has been purified from isolated cytochrome b-c1 complex to a single polypeptide, using a procedure involving Triton X-100 and urea solubilization, calcium phosphate column chromatography and ammonium sulfate fractionation. The purified protein contains 30 nmoles heme per mg protein and has an apparent molecular weight of 30,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. Bacterial cytochrome c1 is soluble in aqueous solution in the absence of detergent and has spectral characteristics similar to mammalian cytochrome c1. The amino acid compositions of these two proteins, however, are not comparable.     

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Studies on cytochrome c oxidase, VIII. The amino acid sequence of polypeptide VII.

The sequence determination of polypeptide VII from beef heart cytochrome c oxidase is described. The amino acid sequence is deduced from overlapping tryptic peptides and peptides obtained after cleavage with Staphylococcus aureus protease. The protein consists of 85 amino acids corresponding to a Mr of 10026, in agreement with a value of 9500 obtained by sodium dodecyl sulfate gel electrophoresis. The amino acid sequence around the only methionine present is very similar to sequences around the invariant heme binding methionine of the cytochrome c family. This similarity suggests that the protein is one of the heme bindings subunits of the oxidase.     

Characterization of a soybean leaf protein that is related to the seed lectin and is increased with pod removal

Levels of several polypeptides in addition to the vegetative storage protein (VSP) increase in soybean leaves following depodding. Two of these polypeptides interact specifically with antibodies raised against the seed lectins of Phaseolus vulgaris and soybean. The two polypeptides, which had apparent molecular masses of 29,000 daltons and 33,000 daltons, were present in the sink-deprived plants but not in control podded plants and were the subunit polypeptides of a glycoprotein designated lectin-related protein (LRP). Soybean LRP was purified to near homogeneity by a combination of ammonium sulfate precipitation and gel filtration. Dialysis of the resuspended ammonium sulfate precipitate caused LRP to reprecipitate, and LRP was soluble only in the presence of molar NaCl. The native relative molecular mass of LRP was 119,000 daltons, a size consistent with a tetrameric organization of the two polypeptides. LRP precipitated during dialysis in association with a 28,000 dalton polypeptide. The protein coprecipitating with LRP was identified as the dimer of the 28,000 dalton subunit of VSP, one of three native isomeric forms of VSP occurring in leaves of depodded plants. Although the specific association between LRP and VSP was intriguing, an in vivo interaction between LRP and VSP was doubtful. LRP was shown to be immunologically similar to soybean agglutinin but did not have detectable hemagglutinating activity. LRP also was shown to be made up of polypeptides distinct from soybean agglutinin.     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.     

Cytochrome c1 complexes.

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

Controlled digestion with trypsin as a structural probe for the N-terminal peptide of soluble and membranous cytochrome c.

When purified bovine cytochrome c1 is digested with trypsin under controlled conditions, the heme polypeptide is preferentially converted from a species of molecular weight 30,600 to a heme polypeptide of molecular weight 29,000. The trypsin sensitive peptide bond is located in the N-terminal region of the cytochrome. Both the reduced and oxidized cytochrome are susceptible to hydrolysis by trypsin at the same locus, but the reduced cytochrome is cleaved at an initial rate approximately twofold greater than the oxidized cytochrome. Membranous cytochrome c1, as occurring in cytochrome b-c1 complex or succinate-cytochrome c reductase complex, is not susceptible to trypsin proteolysis under similar conditions, nor after more extensive treatment of the membranes with trypsin, in spite of the fact that cytochrome c1 presumably comes into contact with cytochrome c at the membrane surface during electron transport. These findings are consistent with a model for the structure of cytochrome c1 in situ in which the cytochrome is an integral membrane protein, located primarily in the membrane continuum, while still having the heme-containing portion of the protein available at the membrane surface for electron transfer to cytochrome c.     

Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays

Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c₁ subunits and by inference from comigration.