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PubMed1998

Mechanism of ubiquinol oxidation by the cytochrome bc1 complex: pre-steady-state kinetics of cytochrome bc1 complexes containing site-directed mutants of the Rieske iron-sulfur protein.

To facilitate characterization of mutated cytochrome bc1 complexes in S. cerevisiae we have developed a new approach using a rapid scanning monochromator to examine pre-steady-state reduction of the enzyme with menaquinol. The RSM records optical spectra of cytochromes b and c1 at 1-ms intervals after a dead time of 2 ms, and menaquinol fully reduces both cytochromes bH and c1 and a portion of cytochrome bL. The rapid-mixing, rapid-scanning monochromator methodology obviates limitations inherent in previous rapid kinetics methods and permits measurements of rates exceeding 200 s-1. To document the validity of this methodology we have examined the reduction kinetics of the cytochrome bc1 complexes from wild-type yeast and yeast that lack ubiquinone. The results establish that menaquinol reacts via the Q cycle pathway both in the presence and absence of ubiquinone. From analyzing bc1 complexes containing Rieske proteins in which the midpoint potential of the iron-sulfur cluster has been altered from +280 to +105 mV, we propose a mechanism in which the protonated quinol displaces a proton from the imidazole nitrogen of one of the histidines that is a ligand to the iron-sulfur cluster and forms a quinol-imidazolate complex that is the electron donor to the redox active iron.

Animals↗
PubMed1996

Antimycin resistance and ubiquinol cytochrome c reductase instability associated with a human cytochrome b mutation.

Progressive exercise intolerance was associated with a decreased maximal rate of ubiquinol cytochrome c reductase (complex III) activity in the muscle mitochondria of the studied patient and with a thirty five-fold increase in the I50 for antimycin A. In contrast, myxothiazol sensitivity was not altered. Complex III activity was stable at 37 degrees C, but progressively decreased at 4 degrees C. An heteroplasmic G to A mutation at position 15615 of the mitochondrial DNA, resulting in the replacement of the highly conserved Gly290 in cytochrome b by Asp, was identified. Histochemical studies showed increased cytochrome oxidase and succinate dehydrogenase activities under the sarcolemma of type I fibres. After partial extraction of mitochondria from the muscle, the residual pellet contained a lower percentage of the mutation than did whole muscle, suggesting that the percentage of mutation is higher in the most readily extracted mitochondria, most probably present under the sarcolemma. In the current 8 transmembrane helix model of cytochrome b, Gly290 lies at the end of the sixth transmembrane helix, facing the intermembrane space and close to the presumed sites of interaction between cytochrome b, the iron-sulfur protein and the 9.5 kDa protein. Since immunoblotting experiments showed a relative decrease in the proportions of these three subunits in the patient's mitochondria compared with the other complex III subunits, it is probable that the complex III instability and the relative decrease in these subunits are related to the mutation. The relationship between the decrease in the apparent affinity for antimycin A and the instability of complex III are discussed.

Amino Acid Sequence↗
PubMed2001

C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

Amino Acid Sequence↗
PubMed1990

The rat cytochrome c oxidase subunit IV gene family: tissue-specific and hormonal differences in subunit IV and cytochrome c mRNA expression.

We have isolated three members of the rat cytochrome c oxidase subunit IV gene family: one functional gene and two processed pseudogenes. The pseudogenes appear to represent the only other closely related sequences in this family. The functional gene encodes an isoform which is expressed in all tissues examined and has features characteristic of 'housekeeping' genes. These include multiple transcription start sites mapped to within an approximately 50 bp region and a GC-rich promoter lacking typical CCAAT or TATAA sequences. Although the subunit IV gene is expressed at its highest levels in cardiac and skeletal muscle, consistent with the high energy demand in those tissues, its expression differs from that of cytochrome c in several respects. 1) Subunit IV mRNA abundance in various tissues is relatively uniform when compared to the highly variable levels of cytochrome c mRNAs. 2) Unlike cytochrome c, subunit IV mRNA is expressed at a surprisingly high level in testis. 3) While cytochrome c mRNA levels in liver are increased markedly in response to thyroid hormone treatment, subunit IV mRNA is not significantly affected. Differences in the expression of these two nuclear-encoded respiratory genes are consistent with differences in regulatory elements within their promoters. Therefore, the regulation of nuclear-encoded respiratory genes in response to tissue demands for cellular energy may not be satisfactorily explained by a set of universal regulators common to all such genes.

Animals↗
PubMed1987

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum.

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b(5) and NADH-cytochrome b(5) reductase from the microsomal fraction of Pisum sativum. The cytochrome b(5) component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b(5) component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.

Journal Article↗
PubMed1979

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. Mitochondrial protein synthesis and analysis of the polypeptide composition of cytochrome c oxidase.

Three previously isolated mutants of Neurospora crassa, temperature-sensitive for the production of cytochrome aa3, have been further analyzed. These mutants have a slightly reduced capacity for mitochondrial protein synthesis when grown at 41 degrees C, although this relative deficiency appeared to be no greater than the deficiency in other cytochrome-aa3-deficient mutants. Thermolability studies revealed that the cytochrome c oxidase purified from each of the mutants grown at 23 degrees C is no more sensitive to heat inactivation than the enzyme isolated from wild-type cells. Sodium dodecylsulfate gel electrophoresis of immunoprecipitates obtained from the mitochondria of each of the mutants grown at 23 degrees C, using antiserum directed against holocytochrome c oxidase, indicated that all the subunits of cytochrome c oxidase were present in relative amounts similar to those found in mitochondria from wild-type cultures. However, when the mitochondria from mutant cultures grown at 41 degrees C were examined in the above fashion, only subunits 5 and 6 of the oxidase were detected. Nonetheless, the mitochondrially synthesized subunit 1, 2 and 3 polypeptides could be immunoprecipitated from mitochondria isolated from mutant cells grown at 41 degrees C and labelled with [3H]leucine in medium containing cycloheximide. Although subunits 4 and 7 could not be detected, because a suitable antibody was not available, the fact that five of the seven subunits were present, but not associated with each other, suggested that the genetic defects in these mutants may affect the process of cytochrome c oxidase assembly.

Electron Transport Complex IV↗
PubMed1993

Reduction of nitro and azo compounds by NADPH-cytochrome P-450 reductase-cytochrome c heme peptide system.

Octa heme peptide, an enzymic digestion product of Candida Krusei cytochrome c, was found to catalyze nitro and azo reduction in the presence of NADPH and NADPH-cytochrome P-450 reductase under anerobic conditions. The reduction was dependent on the concentrations of heme peptide and reductase, and inhibited by carbon monoxide and oxygen. Comparison of the activities of the reductase-heme peptide system with liver microsomes revealed that heme peptide was as effective as cytochrome P-450 in nitro reduction, although less effective in azo reduction. Thus, cytochrome c heme peptide may serve as an artificial substitute for cytochrome P-450 at least in the reductive metabolism of xenobiotics.

Amino Acid Sequence↗
PubMed1978

Electronic state of heme in cytochrome oxidase III. The magnetic susceptibility of beef heart cytochrome oxidase and some of its derivatives from 7-200 K. Direct evidence for an antiferromagnetically coupled Fe (III)/Cu (II) pair.

The temperature dependence of the paramagnetic susceptibility of cytochrome oxidase and some of its derivatives has been measured from 7 to 200 K. The results obtained for the fully oxidized (resting) enzyme correspond exactly to the requirements of the model recently proposed by Palmer et al. (Palmer, G., Babcock, G. T., and Vickery, L. E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210) in which the enzyme possesses two magnetically isolated spin S = 1/2 centers and a spin-coupled S = 2 center. The S = 2 center paramagnetism has been interpreted as arising from a [cytochrome a33+(S = 5/2)--Cuu2+(S = 1/2)] antiferromagnetically coupled iron.copper binuclear complex of total spin S = 2 with -J greater than or equal to 200 cm-1. In addition, the wide temperature range used in the present studies has permitted an analysis of present and other available data (T less than 4K measurements) which readily accommodates results from this and other laboratories (Moss, T.H., Shapiro, E., King, T.E., Beinert, H., and Hartzell, C. R. (1978) J. Biol. Chem 253, 8072-8073) so that a fully consistent picture of the magnetic centers in cytochrome oxidase now appears to be available. Furthermore, anomalous magnetic behavior for the oxidized enzyme.cyanide complex has been interpreted in terms of an antiferromagnetic exchange interaction operating in the binuclear complex [cytochrome a33+.CN-(S = 1/2)--Cuu2+(S = 1/2)] with -J congruent to 40 cm-1. A structural model for the [cytochrome a3(3+)-bridge-CUu2+] center is advanced in which an imidazolate ion serves as the bridging ligand in a manner similar to that found in superoxide dismutase.

Animals↗
PubMed1990

Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction.

Time-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a2+(3)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 v(Fe2(+)-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency we measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/CuA pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a3+(3), with t1/2 congruent to 200 microseconds.

Animals↗
PubMed1985

Independent lateral diffusion of cytochrome bc1 complex and cytochrome oxidase in the mitochondrial inner membrane.

Distinct fluorophores have been conjugated to antibodies for cytochrome bc1 complex and cytochrome oxidase, two integral electron transferring proteins in the mitochondrial inner membrane. Addition of these fluorescent antibodies to preparations of mitochondrial inner membranes followed by appropriate secondary antibodies causes distinct and independent aggregation of the two cytochrome proteins. These results reveal that both cytochrome bc1 complex and cytochrome oxidase diffuse laterally in the membrane plane independent of one another consistent with the random collision model for electron transport in the mitochondrial inner membrane.

Animals↗
PubMed1983

[Reaction of oxidized cytochrome oxidase with cyanide. Effects of pH, cytochrome c and membrane environment].

Binding of HCN with ferric beef heart cytochrome oxidase has been studied in submitochondrial particles, as with the enzyme solubilized in detergent or reconstituted into proteoliposomes. Under all conditions, the reaction proceeds via an intermediate and its kinetics can be described by formal parameters Km and kmax in keeping with the Michaelis-type equation. Km of the reaction strongly depends on the enzyme environment; thus it increases 100-1000 fold upon solubilization of cytochrome oxidase but can be subsequently decreased by incorporation of the enzyme in liposomes and by addition of cytochrome c. pH-dependence of the reaction rate shows that, in submitochondrial particles and proteoliposomes as well as in the case of solubilized enzyme supplement with cytochrome c, HCN specifically binds the form of cytochrome oxidase in which a heme-linked ionizable group with pKa 6,5-6,9 is protonated.

Animals↗
PubMed1981

Cytochrome c oxidase in cytochrome c oxidase-deficient mutant strains of Neurospora crassa.

Three mutant strains of Neurospora crassa, previously characterized as cytochrome c oxidase deficient, were found to possess this respiratory enzyme. The mitochondrial cytochrome contents of logarithmic phase cells of wild type (74A) and the respiration-deficient strains mi-3, cya-4-23, and cya-5-34 were investigated with low temperature difference spectrophotometry; two of the strains were found to possess cytochrome a. Cytochrome c oxidase catalytic activity measurements performed on isolated mitochondria showed that all three mutant strains contained significant amounts of active enzyme which was subsequently found to be active in vivo. However, activity of the cyanide-insensitive, alternate electron transport pathway was responsible for most of the oxygen consumed by the logarithmic phase cells of these mutant strains. Electrophoretic analysis of the polypeptide subunits of cytochrome c oxidase radiolabeled in vivo during logarithmic phase growth showed that, in contrast to previous reports, the respiration-deficient strains appeared to contain all seven subunits of the enzyme.

Electron Transport Complex IV↗
PubMed1982

Mössbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus.

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with Mössbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with Mössbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The Mössbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.

Cyanides↗
PubMed1984

The nuclear-coded subunits of yeast cytochrome c oxidase. III. Identification of homologous subunits in yeast, bovine heart, and Neurospora crassa cytochrome c oxidases.

Sequences for the NH2-terminal halves of subunits IV, V, VI, VII, and VIIa from yeast cytochrome c oxidase have been determined and used to identify homologous subunits in bovine heart and Neurospora crassa cytochrome c oxidases. In conjunction with the complete sequence of subunit VIII (S. D. Power, M. A. Lochrie , T. E. Patterson, and R. O. Poyton (1984) J. Biol. Chem. 259, 6571-6574), we have been able to identify counterparts to yeast subunits IV, V, VI, and VIII in bovine heart cytochrome c oxidase and counterparts to yeast subunits IV and V in Neurospora crassa cytochrome c oxidase. The sequences of these nuclear-coded subunits are conserved between species at a level of 30-50%. Thus, they are conserved to the same extent as the three mitochondrially coded subunits (I, II, and III). The similar degree of homology between species for both the nuclear and mitochondrially coded subunits of cytochrome c oxidase suggests that both sets of polypeptides are conserved coordinately and are, therefore, important components of the functional holoenzyme.

Amino Acid Sequence↗
PubMed1997

Reconstruction of mitochondrial cytochrome P450-dependent steroid hydroxylases in artificial phospholipid membranes: studies of cytochrome P450scc topology by limited proteolysis.

Highly purified cytochrome P450scc from bovine adrenal cortex mitochondria was inserted in artificial phospholipid membranes prepared from phosphatidylcholine to study the main principles of its membrane organization in the model system. Topology of the cytochrome P450scc polypeptide chain in proteoliposomes was studied by limited proteolysis with trypsin or chymotrypsin followed by immunochemical identification of the products of proteolysis products of the membrane-bound heme protein. It is shown that limited proteolysis of cytochrome P450scc in proteoliposomes results in a significant decrease of Vmax for the reaction of cholesterol hydroxylation to pregnenolone in the reconstituted system in the presence of exogenously added adrenodoxin-reductase and adrenodoxin. However, after proteolytic modification of cytochrome P450scc with trypsin and chymotrypsin the affinity of the heme protein to adrenodoxin is increased. Different models of membrane organization as well as functional specificity of cytochrome P450scc in artificial membranes are discussed.

Adrenal Cortex↗
PubMed1994

Differential roles of Glu318 and Thr319 in cytochrome P450 1A2 catalysis supported by NADPH-cytochrome P450 reductase and tert-butyl hydroperoxide.

The kinetic values for 7-ethoxycoumarin (7-EC) hydroxylation have been obtained in both the NADPH-cytochrome P450 reductase- and tert-butyl hydroperoxide (TBHP)-supported systems for several Glu318 and Thr319 mutants of cytochrome P450 1A2. The results with the reductase-supported system suggest that Glu318 is important for both substrate binding and catalysis, whereas Thr319 is critical for neither, although the size of the residue at position 319 influences catalytic activity. In contrast, neither Glu318 nor Thr319 appears to be important for catalytic turnover in the TBHP-supported system despite the fact that the size of the amino acid at position 319 affects the binding of TBHP and 7-EC in opposite manners. The roles of these two distal amino acids in the cytochrome P450 1A2-catalyzed oxidation of 7-EC therefore differ for the reactions supported by cytochrome P450 reductase and TBHP.

Amino Acid Sequence↗
PubMed1997

Anion binding to cytochrome c2: implications on protein-Ion interactions in class I cytochromes c.

The binding of several inorganic and carboxylate anions to cytochrome c2 from Rhodopseudomonas palustris has been investigated by monitoring the salt-induced changes in the redox potential of the heme, using an interpretative model based on the extended Debye-Hückel equation. Most anions were found to interact specifically with the protein at one or multiple sites. Binding constants to the oxidized protein in the range 10(1)-10(2) m-1 were determined from the anion concentration dependence of the chemical shift of the isotropically shifted heme methyl resonances. For several anions the stoichiometry and strength of the binding to cytochrome c2 were found comparable with those determined for mitochondrial cytochromes c, in spite of the limited sequence similarity (less than 40%) and the lower positive charge of the bacterial protein. These analogies were interpreted as indicative of the existence of common binding sites which are proposed to be located in the conserved lysine-rich domain around the solvent-exposed heme edge, which is also the surface area likely involved in the interaction with redox partners. The changes in E degrees due to partial neutralization of the positive charge of cytochrome c2 due to specific anion binding were found comparable with those for the mitochondrial species.

Anions↗
PubMed2001

Expression, purification, and physical properties of recombinant flavocytochrome fusion proteins containing rat cytochrome b(5) linked to NADPH-cytochrome P450 reductase by different membrane-binding segments.

Reconstitution of the enzymatic activities using purified microsomal cytochrome P450s (P450) requires the presence of a membrane-binding segment in the mammalian flavoprotein, NADPH--cytochrome P450 reductase (CPR), and the hemeprotein, cytochrome b(5) (b(5)). The mechanism(s) by which the membrane-binding segments of these proteins exert such a critical role in influencing the reconstitution of the NADPH-supported activity of a P450 remains undefined. In the present work we describe the construction, expression, and purification of four different types of recombinant flavocytochromes containing rat b(5) and rat CPR linked by various membrane-binding segments. The physical properties of these artificial fusion proteins have been studied to determine their ability to serve as electron transfer agents. These studies are a prelude to the subsequent study (accompanying paper) evaluating the functional roles of the hydrophobic (membrane-binding) sequences of b(5) and CPR in the reconstitution of P450 activities. The present study shows that the purified recombinant fusion proteins can serve as active electron transport carriers from NADPH to cytochrome c as well as b(5) by intramolecular as well as intermolecular reactions. It is shown here that the electron transport properties of these purified fusion proteins are influenced by high concentrations of KCl, suggesting a role for charged amino acids in protein-protein interactions. The present study illustrates the application of artificial recombinant flavocytochromes as useful proteins for the study of intramolecular electron transport reactions for comparison with intermolecular interactions.

Animals↗