Stabilization of cellular mitochondrial enzyme complex and sialyltransferase activity through supplementation of 30Kc19 protein.

In previous studies, 30Kc19, a lipoprotein in silkworm hemolymph, enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro. In this study, we hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme. We investigated this using in vitro and cellular assessments. The activity of sialyltransferase (ST) and isolated mitochondrial complex I/III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, ST activity inside erythropoietin (EPO)-producing Chinese hamster ovary (CHO) cells increased more than 25 % and mitochondrial complex II activity in HeLa cells increased more than 50 % with 30Kc19. The increase in intracellular ST activity resulted in an increase in sialic acid content of glycoproteins produced in CHO cells supplemented with 30Kc19. Similarly, enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19 by over 50 %. Because 30Kc19 stabilized intracellular enzymes for glycosylation and enhanced protein productivity with supplementation in the culture medium, we expect that 30Kc19 can be a valuable tool for effective industrial recombinant protein production.

Fertility-related proteomic profiling bull spermatozoa separated by percoll.

Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H+ transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.

Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix.

BACKGROUND: The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species. RESULTS: Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectrum of the isolated c-type cytochrome showed a characteristic α-band peak at 553 nm corresponding to heme C. The pyridine hemochrome spectrum also revealed the presence of heme B. In non-denaturing polyacrylamide gel electrophoresis, the cytochrome migrated as a single band with an apparent molecular mass of 80 kDa, and successive SDS-PAGE separated the 80-kDa band into 3 polypeptides with apparent molecular masses of 40, 30, and 25 kDa. The results of mass spectrometry indicated that the 25-kDa band corresponded to the hypothetical cytochrome c subunit encoded by the ORF APE_1719.1. In addition, the c-type cytochrome-containing polypeptide complex exhibited menaquinone: yeast cytochrome c oxidoreductase activities. CONCLUSION: In conclusion, we showed that A. pernix, a hyperthemophilic archaeon, has a "full" bc complex that includes a c-type cytochrome, and to the best of our knowledge, A. pernix is the first archaea from which such a bc complex has been identified. However, an electron donor candidates for cytochrome c oxidase, such as a blue copper protein, have not yet been identified in the whole genome data of this archaeon. We are currently trying to identify an authentic substrate between a bc complex and terminal oxidase.

Purification of Saccharomyces cerevisiae mitochondria by zone electrophoresis in a free flow device.

This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, 2-DE or electron microscopy.

Organization of assembly factors Cbp3p and Cbp4p and their effect on bc(1) complex assembly in Saccharomyces cerevisiae.

The bc(1) complex (complex III) of Saccharomyces cerevisae is composed of ten subunits that are assembled in the inner mitochondrial membrane. Cbp3p and Cbp4p are two mitochondrial proteins which are postulated to act as chaperones in bc(1) complex formation. Here, we show by blue native PAGE that cbp3Delta and cbp4Delta mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover, deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high-molecular-weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high-level expression of Cbp3p can partially compensate for the lack of Cbp4p. The finding that mitochondria of cbp3Delta and cbp4Delta mutants exhibit a wild-type lipid composition favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism.

Interaction of an anticancer ruthenium complex HInd[RuInd2Cl4] with cytochrome c.

Cytochrome c is an important electron transfer protein in the respiratory chain, shuttling electrons from cytochrome c reductase to cytochrome c oxidase. Extensive chemical modification studies indicate significant electrostatic interactions between these proteins and show that all structural and conformational changes of cytochrome c can influence the electron transport. In the present work we examine the effect of an anticancer ruthenium complex, trans-Indazolium (bisindazole) tetrachlororuthenate(III) (HInd[RuInd(2)Cl(4)]), on the conformation of cytochrome c, the state of the heme moiety, formation of the protein dimer and on the folding state of apocytochrome c. For this purpose, gel-filtration chromatography, absorption second derivative spectroscopy, circular dichroism (CD) and inductively coupled plasma atomic emission spectroscopy (ICP(AES)) were used. The present data have revealed that binding of the potential anticancer drug HInd[RuInd(2)Cl(4)] complex to cytochrome c induces a conformation of the protein with less organized secondary and tertiary structure.

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase.

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.

Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain.

Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity [Obungu et al. (2000) Biochim. Biophys. Acta 1457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.

Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment.

BACKGROUND: The cytochrome bc(1) complex is part of the energy conversion machinery of the respiratory and photosynthetic electron transfer chains. This integral membrane protein complex catalyzes electron transfer from ubiquinol to cytochrome c. It couples the electron transfer to the electrogenic translocation of protons across the membrane via a so-called Q cycle mechanism. RESULTS: The cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae was crystallized together with a bound antibody Fv fragment. The structure was determined at 2.3 A resolution using multiple isomorphous replacement, and refined to a crystallographic R factor of 22.2% (R(free) = 25.4%). The complex is present as a homodimer. Each 'monomer' of the refined model includes 2178 amino acid residues of subunits COR1, QCR2, COB, CYT1, RIP1, QCR6, QCR7, QCR8 and QCR9 of the cytochrome bc(1) complex and of the polypeptides V(H) and V(L) of the Fv fragment, the cofactors heme b(H), heme b(L), heme c(1), the [2Fe-2S] cluster and 346 water molecules. The Fv fragment binds to the extrinsic domain of the [2Fe-2S] Rieske protein and is essential for formation of the crystal lattice. CONCLUSIONS: The approach to crystallize membrane proteins as complexes with specific antibody fragments appears to be of general importance. The structure of the yeast cytochrome bc(1) complex reveals in detail the binding sites of the natural substrate coenzyme Q6 and the inhibitor stigmatellin. Buried water molecules close to the binding sites suggest possible pathways for proton uptake and release. A comparison with other cytochrome bc(1) complexes shows features that are specific to yeast.

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides.

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.

Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: characterization of the iron-sulfur centers from the dehydrogenases and investigation of the high-potential iron-sulfur protein function by in vitro reconstitution of the respiratory chain.

Rhodothermus marinus, a thermohalophilic bacterium, has a unique electron-transfer chain, containing, besides a cbb3 and a caa3 terminal oxidases, a novel cytochrome bc complex [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1268-1275]. The membrane-bound iron-sulfur centers of this bacterium were studied by electron paramagnetic resonance (EPR) spectroscopy, leading to the identification of its main electron-transfer complexes. The resonances typical for the Rieske-type centers are not detected. Clusters S1 and S3 from succinate dehydrogenase were identified; interestingly, center S3 is shown to be present in two different conformations, with g values at 2.035, 2.009, and 2.001 and at 2.025, 2.002, and 2.000. Upon addition of NADH and dithionite, EPR signals assigned to resonances characteristic of binuclear and tetranuclear clusters develop and are attributed to the iron-sulfur centers of complexes I and II. A high-potential iron-sulfur protein- (HiPIP-) type center previously detected in the membranes of this bacterium [Pereira et al. (1994) FEBS Lett. 352, 327-330] is shown to belong indeed to a canonical HiPIP. This protein was purified and extensively characterized. It is a small water-soluble protein of approximately 10 kDa, containing a single [4Fe-4S]3+/2+ cluster. The reduction potential, determined by EPR redox titrations in intact and detergent-solubilized membranes as well as by cyclic voltammetry in solution, has a pH-independent value of 260 +/- 20 mV, in the range 6-9. In vitro reconstitution of the R. marinus electron-transfer chain shows that the HiPIP plays a fundamental role in the chain, as the electron shuttle between R. marinus cytochrome bc complex and the caa3 terminal oxidase, being thus simultaneously identified a HiPIP reductase and a HiPIP oxidase.

Electron transfer proteins from the haloalkaliphilic archaeon Natronobacterium pharaonis: possible components of the respiratory chain include cytochrome bc and a terminal oxidase cytochrome ba3.

Natronobacterium pharaonis, an aerobic haloalkaliphilic archaebacterium, expresses high concentrations of redox proteins as do alkaliphilic eubacteria. The first redox protein characterized from N. pharaonis was halocyanin [Scharf, B., & Engelhard, M. (1993) Biochemistry 32, 12894-12900], a small blue copper protein. It is a peripheral membrane protein and is conjectured to function in a manner similar to plastocyanin. In the present work, the respiratory chain is further elucidated and the purification and characterization of the most abundant components cytochrome bc and cytochrome ba3 from the membrane fraction are described. The cytochrome bc complex consists of a 14 and an 18 kDa subunit in a 1:1 ratio, with heme c bound to the larger polypeptide. An Fe-S subunit similar to that found in eukaryotic bc complexes has not yet been identified. The second membrane complex carries two different heme groups of the ba3-type as well as copper. It contains two subunits of 36 and 40 kDa. This cytochrome ba3 binds carbon monoxide, a feature common to terminal oxidases. There is no spectroscopic evidence for a second terminal oxidase; hence, under the growth conditions chosen the respiratory chain of N. pharaonis appears to be unbranched. In addition to these cytochromes, a succinate dehydrogenase which is solubilized from the membrane by detergents was isolated. A cytochrome c which was isolated from the cytosol has an unusually high molecular weight and a redox potential of -142 mV. A second cytosolic protein, ferredoxin, was purified to homogeneity. A comparison of the redox potentials of the isolated proteins with those obtained from the native membrane allows the construction of a possible electron transfer chain.

Large-scale chromatographic purification of F1F0-ATPase and complex I from bovine heart mitochondria.

A new chromatographic procedure has been developed for the isolation of F1F0-ATPase and NADH:ubiquinone oxidoreductase (complex I) from a single batch of bovine heart mitochondria. The method employed dodecyl beta-delta-maltoside, a monodisperse, homogeneous detergent in which many respiratory complexes exhibit high activity, for solubilization and subsequent purification by ammonium sulphate fractionation and column chromatography. A combination of anion-exchange, gel-filtration, and dye-ligand affinity chromatography was used to purify both complexes to homogeneity. The F1F0-ATPase preparation contains only the 16 known subunits of the enzyme. It has oligomycin-sensitive ATP hydrolysis activity and, as demonstrated elsewhere, when reconstituted into lipid vesicles it is capable of ATP-dependent proton pumping and of ATP synthesis driven by a proton gradient [Groth and Walker (1996) Biochem. J. 318, 351-357]. The complex I preparation contains all of the subunits identified in other preparations of the enzyme, and has rotenone-sensitive NADH:ubiquinone oxidoreductase and NADH:ferricyanide oxidoreductase activities. The procedure is rapid and reproducible, yielding 50-80 mg of purified F1F0-ATPase and 20-40 mg of purified complex I from 1 g of mitochondrial membranes. Both preparations are devoid of phospholipids, and gel filtration and dynamic light scattering experiments indicate that they are monodisperse. Therefore, the preparations fulfil important prerequisites for structural analysis.

Identification, cDNA sequence and deduced amino acid sequence of the mitochondrial Rieske iron-sulfur protein from the green alga Chlamydomonas reinhardtii. Implications for protein targeting and subunit interaction.

Specific oligonucleotide probes were used to isolate a cDNA clone for the mitochondrial Rieske iron-sulfur protein of the green alga Chlamydomonas reinhardtii. The protein is synthesized as a longer precursor with a cleavable N-terminal presequence of 54 amino acids but without a C-terminal extension. Comparison of the predicted secondary structure of this N-terminal sequence with that of the targeting signal of the chloroplast Rieske protein from C. reinhardtii [de Vitry (1994) J. Biol. Chem. 269, 7603-7609] indicates that, although they both have the potential to form amphiphilic alpha helices, the mito-chondrial presequence may form a more hydrophobic helix that could penetrate deeper into the membrane. The N-terminal part of the mature mitochondrial Rieske protein is characterized by a long, strongly hydrophilic N-terminal domain and by a positive charge in the middle of the hydrophobic stretch that is presumed to interact with the bc1 complex. Thus, the protein from C. reinhardtii differs from the Rieske proteins from mammals or fungi.

Membrane association of the Rieske iron-sulfur protein.

The mode of membrane attachment of the Rieske iron-sulfur proteins from cytochrome b6f complex of pea thylakoids and from cytochrome bc1 complex of yeast mitochondria has been studied using biochemical approaches. The relative sensitivity of the Rieske protein to trypsin in the thylakoid membrane shows that all trypsin sites of the Rieske protein are on the lumen side of the thylakoid membrane. In contrast to cytochrome f the chloroplast Rieske protein was extracted from thylakoids using chaotropic agents (NaSCN, urea), an alkaline pH and relatively low concentrations of Trinon X-100. The cytochrome bc1 complex Rieske protein from mitochondrial membranes of yeast was also released by NaSCN and alkaline treatment. The results presented here led us to the conclusion that the mitochondrial and chloroplast Rieske proteins are extrinsic and that their association with the rest of the complex involves hydrophobic interactions.

A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c1.

The alga Polytomella spp. offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall. In this work the mitochondrial bc1 complex from Polytomella spp. was solubilized and purified by ion exchange chromatography. The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol. The molecular mass of the bc1 complex from Polytomella spp. was assayed by gel filtration and estimated to be of 256,300 Da. Therefore, this complex exhibits the unique property of behaving as a monomer. Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed. A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes. With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced. The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH. The data suggest that the cytochrome c1 from Polytomella spp. differs from other protoctists like Crithidia and Euglena, i.e. it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes.

Subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex is required for insertion of EPR-detectable iron-sulfur cluster into the Rieske iron-sulfur protein.

Deletion of QCR9, the nuclear gene encoding the 7.3-kDa subunit 9 of the cytochrome bc1 complex, impairs respiration of Saccharomyces cerevisiae, coincident with loss of ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from yeast in which the gene for subunit 9 is deleted show a diminution of cytochrome b absorption similar to the spectra of membranes from yeast in which the gene for the Rieske iron-sulfur protein is deleted, suggesting an interaction between subunit 9, iron-sulfur protein, and cytochrome b. Synthesis of cytochrome b by mitochondria from the deletion strain is unimpaired, indicating that the diminished b absorption is due to a post-assembly effect on the heme environment resulting from the absence of subunit 9. Iron-sulfur protein is present in normal amounts and processed to its mature form in the absence of subunit 9, although the protein is more labile to endogenous proteases during the isolation of membranes. EPR spectroscopy of membranes from the subunit 9 deletion strain indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature sized apoprotein is present. Pre-steady state reduction of cytochrome c1 is markedly slowed, but not eliminated, in the subunit 9 deletion strain, which suggests that an EPR-silent, sluggishly reactive derivative of the iron-sulfur cluster is present. These results suggest that in the absence of subunit 9 the conformation of iron-sulfur protein is altered such that the protein is more labile, the iron-sulfur cluster is not properly inserted, and iron-sulfur protein interaction with cytochrome b is modified in a manner which distorts the heme environment. This is the first instance in which deletion of one of the supernumerary subunits of the cytochrome bc1 complex results in the loss of function of a redox center within the complex, without a concomitant loss of other subunits.

Abundance, subunit composition, redox properties, and catalytic activity of the cytochrome bc1 complex from alkaliphilic and halophilic, photosynthetic members of the family Ectothiorhodospiraceae.

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy. The gy values of the Rieske [2Fe-2S] clusters observed in membranes of E. mobilis and E. halophila were 1.895 and 1.910, respectively. In E. mobilis membranes, the cytochrome bc1 complex was present in a stoichiometry of approximately 0.2 per reaction center. This complex was isolated and characterized. It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; Em = 116 mV), cytochrome c1 (Em = 341 mV), and a Rieske iron-sulfur cluster. The absorbance spectrum of cytochrome bL displayed an asymmetric alpha-band with a maximum at 564 nm and a shoulder at 559 nm. The alpha bands of cytochrome bH and cytochrome c1 peaked at 559.5 and 553 nm, respectively. These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively. No evidence for the presence of a fourth subunit was obtained. Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced.(mol of cytochrome c1)-1.s-1. The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc1 complexes.

Hydroubiquinone-cytochrome c2 oxidoreductase from Rhodobacter capsulatus: definition of a minimal, functional isolated preparation.

The hydroubiquinone-cytochrome c2 oxidoreductase (cyt bc1) from Rhodobacter capsulatus has been solubilized according to the dodecyl maltoside method and isolated, and its minimal functional composition has been characterized. We find the complex to be composed of three protein subunits corresponding to polypeptides of cyt b (44 kDa), cyt c1 (33 kDa), and 2Fe2S cluster (24 kDa). A fourth band sometimes discernable at 22 kDa appears to be an artifact of the polyacrylamide gel electrophoresis procedure. Its appearance is shown to be derived from the 2Fe2S cluster subunit by the similarity of the binding of subunit-specific monoclonal antibodies and the identical N-terminal sequence of the 24- and 22-kDa bands. The cofactors of cyt bc1, namely, cyt bH, cyt bL, cyt c1, and the 2Fe2S center, the Qos and Qow domains of the Qo site, and the Qi site appear intact as indicated by their optical and EPR spectral signatures, redox properties, and inhibitor binding. The electron paramagnetic resonance spectrum of the cyt bH heme is altered by antimycin, consistent with a change in the dihedral angle between the ligating histidine imidazoles, while the spectrum of the cyt bL heme is broadened by stigmatellin. The ubiquinone-10 content is variable, ranging from 0.8 to 3 molecules/cyt bc1. Activity studies define this three-subunit cyt bc1 complex as a minimal structure, equipped as the enzyme in the native state and capable of full catalytic activity.