Heme Group Of Cytochrome Research Articles

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoinduced electron transfer between cytochrome c peroxidase and horse cytochrome c labeled at specific lysines with (dicarboxybipyridine)(bisbipyridine)ruthenium(II)

The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) [Ru(II)] were studied by flash photolysis. All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI. This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI. The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively. The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives. These results suggest that there are two binding orientations for cytochrome c on CMPI. The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer. Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase. A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength. When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed. The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.

The heme groups of cytochrome o from Escherichia coli.

Cytochrome o, one of the two terminal ubiquinol oxidases of Escherichia coli, is structurally and functionally related to cytochrome c oxidase of mitochondria and some bacteria. It has two heme groups, one of which binds CO and forms a binuclear oxygen reaction center with copper. The other heme is unreactive toward ligands, exhibits strong interactions with the binuclear center, and is mainly responsible for the reduced-minus-oxidized alpha band. Protoheme has been thought to be the prosthetic group of b-type cytochromes, including cytochrome o. However, the hemes of cytochrome o are of a different kind, for which we propose the name heme O. Its pyridine hemochrome spectrum is blue-shifted by 4 nm relative to that of protoheme, and chromatographic behavior showed that it is much more hydrophobic than protoheme. Fast atom bombardment mass spectrometry yielded a molecular mass of 839 Da. Heme O is proposed to be a heme A-like molecule, containing a 17-carbon hydroxyethylfarnesyl side chain, but with a methyl residue replacing the formyl group.

Topological studies of monomeric and dimeric cytochrome c oxidase and identification of the copper A site using a fluorescence probe.

Beef heart cytochrome c oxidase was labeled at a single sulfhydryl group by treatment with 5 mM N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS) at pH 8.0 for 4 h. Sodium dodecyl sulfate gel electrophoresis revealed that the enzyme was exclusively labeled at subunit III, presumably at Cys-115. The high affinity phase of the electron transfer reaction with horse cytochrome c was not affected by acetylamidoethyl-1-aminonaphthalene-5-sulfonate (AEDANS) labeling. Addition of horse cytochrome c to dimeric AEDANS-cytochrome c oxidase resulted in a 55% decrease in the AEDANS fluorescence due to the formation of a 1:1 complex between the two proteins. Forster energy transfer calculations indicated that the distance from the AEDANS label on subunit III to the heme group of cytochrome c was in the range 26-40 A. In contrast to the results with the dimeric enzyme, the fluorescence of monomeric AEDANS-cytochrome c oxidase was not quenched at all by binding horse heart cytochrome c, indicating that the AEDANS label on subunit III was at least 54 A from the heme group of cytochrome c. These results support a model in which the lysines surrounding the heme crevice of cytochrome c interact with carboxylates on subunit II of one monomer of the cytochrome c oxidase dimer and the back of the molecule is close to subunit III on the other monomer. In order to identify the cysteine residues that ligand copper A, a new procedure was developed to specifically remove copper A from cytochrome c oxidase by incubation with 2-mercaptoethanol followed by gel chromatography. Treatment of the copper A-depleted cytochrome c oxidase preparation with 1,5-I-AEDANS resulted in labeling sulfhydryl groups on subunit II as well as on subunit III. No additional subunits were labeled. This result indicates that the copper A binding site is located at cysteines 196 and/or 200 of subunit II and that removal of copper A exposes these residues for labeling by 1,5-I-AEDANS. Alternative copper A depletion methods involving incubation with bathocuproine sulfonate (Weintraub, S.T., and Wharton, D.C. (1981) J. Biol. Chem. 256, 1669-1676) or p-(hydroxymercuri)benzoate (Li, P.M., Gelles, J., Chan, S.I., Sullivan, R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095) were also investigated. Treatment of these preparations with 1,5-I-AEDANS resulted in labeling cysteine residues on subunits II and III. However, additional sulfhydryl residues on other subunits were also labeled, preventing a definitive assignment of the location of copper A using these depletion procedures.

Flash-induced oxidation of cytochrome b-563 in algae under anaerobic conditions: Effect of dinitrophenylether of iodonitrothymol

Oxidation of cytochrome b-563 and reduction of oxidized plastocyanin were studied under reducing conditions in a mutant of Chlorella sorokiniana submitted to a single actinic flash. The rate of both processes is strongly decreased by addition of dinitrophenylether of iodonitrothymol. The slow increase of the membrane potential (phase b) is also strongly inhibited. Thus dinitrophenylether of iodonitrothymol is an efficient inhibitor of the site of oxidation of cytochrome b by Photosystem I (Q z site) in living algae. These results are consistent with the view that the Q z site can catalyze both cytochrome b-563 reduction and oxidation in a mechanism involving just one heme group of cytochrome b. The rate constant of the inhibitor release is higher than 100 s −1.

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc.

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. Förster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.

Correlations between structural and spectroscopic properties of the high-spin heme protein cytochrome c'.

The cytochromes c' are a class of heme proteins whose native spectroscopic properties have been suggested to represent a quantum mechanical admixture of intermediate-(S = 3/2) and high-(S = 5/2) spin states. Here features of the cytochrome c' heme environment, as revealed by X-ray crystallographic studies of the dimeric cytochrome c' from Rhodospirillum molischianum, are related to the observed spectroscopic properties. The environment of the heme group in cytochrome c' supports the existence of the admixed spin state at neutral pH and suggests that pH-dependent transition to a pure high-spin state at alkaline pH involves deprotonation of the histidine axial ligand to the heme iron.

Cytosolic modulators of activities of microsomal enzyme of cholesterol biosynthesis. Role of a cytosolic protein with properties similar to Z-protein (fatty acid-binding protein).

Rat liver cytosol contains proteins and smaller molecules that affect activities of the microsomal enzymes of cholesterol biosynthesis. One protein (mr = 12,000) has been purified by chromatography, gel filtration, and preparative isoelectric focusing. Properties of the protein on sodium dodecyl sulfate-, and anodic-, and cathodic-disc gel electrophoresis are reported in addition to gel filtration and electrofocusing that were used for purification. By comparison of these properties, amino acid compositions, ligand binding, and the abundance in various tissues the protein appears to be very similar to Z-protein that has been shown by others to be identical with hepatic fatty-acid binding protein. The same properties also appear to be very similar to sterol carrier protein. A cytosolic metabolite, heme, which stimulates 4-methyl sterol oxidase, is bound to the protein during purification. When endogeneous heme is removed, both protein and heme are required for maximal stimulation of microsomal 4-methyl sterol oxidase activity. Hemoglobin produces an equal extent of stimulation presumably by heme group exchange, but the nonexchangeable heme group of cytochrome c is ineffective. Thus, the cytosolic protein may promote uptake and retention of relatively more water-soluble substances into the hydrophobic environment of the membrane-bound enzymes of cholesterol biosynthesis. Z-protein exhibits affinity for an exceptionally wide variety of ligands (e.g. fatty acids, azodyes, organic anions, bile pigments, etc) that may either stimulate or inhibit the microsomal enzymes. Accordingly, with the suggestion that the cytosolic protein is Z-protein, the wide variety of potential interactions of both endogenous metabolites and exogenous xenobiotics may account, in part, for facile modulations of activities of the rate-limiting microsomal synthetic enzymes in various physiological and nutritional states.

Comparative characterisation of nitrate reductase from wild-type and molybdenum cofactor-defective cell cultures of Nicotiana tabacum

Affinity-purified nitrate reductase (EC 1.6.6.1) from wild-type and the molybdenum cofactor-defective cnx-68/2 cell line of Nicotiana tabacum has been comparatively characterised with respect to enzymological properties. K m values and pH optima for the 4 nitrate reductase-associated activities, including diaphorase, are presented. Exposure to different inhibitors and moderate heat (45°C) reveal the tobacco nitrate reductase to be functionally divided into two distinct parts. Both the wild-type and cnx mutant nitrate reductase contain a heme group of cytochrome b type, possess an identical sedimentation coefficient of 7.6S and an identical gel filtration derived molecular weight of 200 000. Both enzymes were found to exhibit similar enzymatic parameters, inhibitor specificities and heat stabilities. Upon affinity chromatography, varying portions of the 7.6S holoenzyme dissociate into slower sedimenting (about 4S) subunits with cytochrome c reductase activity, which has been established for the wild-type as well as the cnx mutant enzyme. From the data obtained it can be concluded that (1) the apoprotein of the cnx mutant nitrate reductase is unaffected by the mutation while the defect of this enzyme type resides exclusively in the molybdenum-cofactor; (2) the cnx mutant nitrate reductase possesses a molybdenum-cofactor, defective in its catalytic properties, but still able to mediate the assembly of the cytochrome c reductase subunits to form the 7.6S holoenzyme.

The orientation of the heme group in crystalline cytochrome b5

The correct orientation of the heme group in cytochrome b 5 has been determined by difference Fourier techniques at 2.0 Å resolution. The heme orientation is now in agreement with that obtained in a recent high resolution NMR study.

Fluorescence studies of cytochrome c1 and a cytochrome c1-cytochrome c complex

Summary Cytochrome c 1 and cytochrome c form a complex in aqueous solution. The complex is stable to chromatography on Sephadex but is dissociated in media of high ionic strength. Results from the fluorescence probe technique using 8-anilino-1-naphthaline sulphonic acid strongly suggest that (i) the heme group of cytochrome c 1 is completely buried in the protein moiety, in contrast to cytochrome c ; (ii) ferricytochrome c 1 undergoes a conformation change on reduction which produces a more tightly closed structure; and (iii) in the cytochrome c -cytochrome c 1 complex the edge of the cytochrome c heme group evidently continues to be exposed.

Nuclear magnetic resonance study of the interaction of cytochrome c with cytochrome c peroxidase

The hyperfine shifted resonances in the nuclear magnetic resonance (NMR) spectrum of cytochrome c broaden upon addition of cytochrome c peroxidase in a manner which indicates 1:1 reversible complex formation between cytochrome c and cytochrome c peroxidase. Since, in the presence of excess cytochrome c, we see a time-averaged NMR spectrum and not a simple superposition of two distinct spectra from free and complexed cytochrome c, the exchange between the two forms is fast on the NMR time scale, yielding for the off-rate of the complex a lower limit of 200/s. (The on-rate is several order of magnitude greater than the off-rate.) The observed line widths of methyl resonances are, consistent with theory, dependent on the rotational correlation time of the whole macromolecule/macromolecular complex. The widths of the heme ring methyls of cytochrome c in cytochrome c-peroxidase complex are not sensitive to the electronic spin state changes in the cytochrome c peroxidase heme iron on addition of cyanide or fluoride showing that the heme groups of cytochrome c and cytochrome c peroxidase are sufficiently far from each other. The unpaired electron spin density distribution over the heme ring of cytochrome c in cytochrome c-peroxidase complex is slightly altered with respect to free cytochrome c as indicated by observed small changes in the positions of the ring methyl resonances. This could arise from a small conformational change in cytochrome c on complexing with cytochrome c peroxidase.

Preparation and properties of cytochrome b 2 from yeast

A simple method, which yields about 10 ml. of 1 × 10 −5 M cytochrome b 2 from 100 g. baker's yeast is described. The hematin iron content of the preparation was 0.025 %. The ratio of flavin mononucleotide to heme groups was one to one. The activity was very high. At 3 × 10 −3 M l(+)-lactate, 5 × 10 −5 M ferricytochrome c, and pH 7.4, about 9000 heme groups of cytochrome c were reduced per minute per heme per flavin group of the preparation. The reaction is first order for ferricytochrome c, and the constant is 3.5 × 10 6/sec./mole enzyme. Lowering the pH reduced the reaction speed. A p K of 5.65 with n = 1 is evident. Half-maximal activity was obtained for 1.85 × 10 −5 M ferricytochrome c and excess lactate, or for 3.5 × 10 −4 M l(+)-lactate and excess ferricytochrome c at pH 7.4. The enzyme is specific for l(+)-lactate. d(−)-Lactate does not inhibit the reaction. Versene activates the reaction. Thiol inhibitors and atebrin exert a powerful inactivation of the preparation.