The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.     

Generation,characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

Cytochrome bc₁ complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c₂) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc₁ complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c₁ with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c₁-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5 Å resolution.     

Preparation and characterization of the water-soluble heme-binding domain of cytochrome c1 from the Rhodobacter sphaeroides bc1 complex.

The ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, cytochrome c1, is the site of interaction with cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the cytochrome c1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the cytochrome c1 subunit that lacks the membrane anchor at the carboxyl terminus. The bc1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of cytochrome c1 in the periplasm. The water-soluble cytochrome c1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble cytochrome c1 is competent as a reductant for cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble cytochrome c1 retains many of the properties of the membrane-bound subunit of the bc1 complex and, therefore, will be useful for further structural and functional characterization.     

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.     

Large-scale purification and characterization of a highly active four-subunit cytochrome bc1 complex from Rhodobacter sphaeroides

A highly active, large-scale preparation of ubiquinol:cytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Rhodobacter sphaeroides. The enzyme was solubilized from chromatophores by using dodecyl maltoside in the presence of glycerol and was purified by anion-exchange and gel filtration chromatography. The procedure yields 35 mg of pure bc1 complex from 4.5 g of membrane protein, and its consistently results in an enzyme preparation that catalyzes the reduction of horse heart cytochrome c with a turnover of 250-350 (mumol of cyt c reduced).(mumol of cyt c1)-1.s-1. The turnover number is at least double that of the best preparation reported in the literature [Ljungdahl, P. O., Pennoyer, J. D., Robertson, D. C., & Trumpower, B. L. (1987) Biochim. Biophys. Acta 891, 227-241]. The scale is increased 25-fold, and the yield is markedly improved by using this protocol. Four polypeptide subunits were observed by SDS-PAGE, with Mr values of 40K, 34K, 24K, and 14K. N-Terminal amino acid sequences were obtained for cytochrome c1, the iron-sulfur protein subunit, and for cytochrome b and were identical with the expected protein sequences deduced from the DNA sequence of the fbc operon, with the exceptions that a 22-residue fragment is processed off of the N-terminus of cytochrome c1 and the N-terminal methionine residue is cleaved off both the b cytochrome and iron-sulfur protein subunits. Western blotting experiments indicate that subunit IV is not a contaminating light-harvesting complex polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit IV (Mr = 14,384) of the cytochrome b-c1 complex from Rhodobacter sphaeroides. Cloning, DNA sequencing, and ubiquinone binding domain.

The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.     

Inhibitor-complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides

The cytochrome bc(1) complex (bc(1)) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(1) and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.     

fbc operon, encoding the Rieske Fe-S protein cytochrome b, and cytochrome c1 apoproteins previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudomonas capsulata

Detailed comparison of the 'Rhodopseudomonas sphaeroides GA' strain used by Gabellini et al. (1985) with genuine R. sphaeroides and R. capsulata strains indicated that the previously reported fbc operon of R. sphaeroides (Gabellini and Sebald, 1986) encoding the structural genes for the Rieske Fe-S protein, cytochrome b and cytochrome c1 subunits of the ubiquinol:cytochrome c2 oxidoreductase, is not from R. sphaeroides, but is rather from a strain of R. capsulata. Consequently, the genuine bc1 genes from R. sphaeroides were cloned using corresponding R. capsulata genes as probes, and a partial nucleotide sequence for the Rieske Fe-S protein of R. sphaeroides was determined and compared with that of R. capsulata.     

Assignment of the histidine axial ligands to the cytochrome bH and cytochrome bL components of the bc1 complex from Rhodobacter sphaeroides by site-directed mutagenesis.

The cytochrome b subunit of the bc1 complex contains two cytochrome components, cytochrome bH and cytochrome bL. Sequence comparisons of this polypeptide from a number of organisms have revealed four invariant histidines which have been postulated to be the heme ligands for the two protoheme IX prosthetic groups. In Rhodobacter sphaeroides, these correspond to His97, His111, His198, and His212. In this paper, the results of amino acid substitutions at each of these positions are reported. Replacement of His97 by either Asp or Asn and of His198 by Asn or Tyr resulted in loss of both cytochrome components. However, His111Asn, His111Asp, and His212Asp all resulted in the selective loss of cytochrome bH and the retention of cytochrome bL. Furthermore, flash kinetics studies show that the myxothiazol-sensitive quinol oxidase (Qz) site associated with cytochrome bL is still functional. These data support the assignment of the axial ligands to cytochrome bH (His111 and His212) and cytochrome bL (His97 and His198). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane alpha-helices.     

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.     

Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150,000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30,000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c1 is more exposed than is that of mammalian cytochrome c1, but less exposed than that of cytochrome c2. Ferricytochrome c1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c1, suggesting that the sulfhydryl groups of cytochrome c1 are the electron donors for photoreduction. Purified cytochrome c1 contains 3 +/- 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c1, the bacterial protein does not form a stable complex with cytochrome c2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c1 and bacterial ferricytochrome c2, and between bacterial ferrocytochrome c1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c1.     

Electrochemical and spectroscopic investigations of the cytochrome bc1 complex from Rhodobacter capsulatus.

The cytochrome bc(1) complex from Rhodobacter capsulatus was investigated by protein electrochemistry and visible/IR spectroscopy. Infrared difference spectra, which represent redox-induced conformational changes of cofactors and their protein environments, show signals of the hemes, the quinone Q(i), and small conformational changes of the protein backbone. Furthermore, band features were tentatively assigned to protonated aspartic or glutamic acids involved in the redox transition of each of the b hemes, a proline in that of the [2Fe-2S] protein, and an arginine in that of cytochrome b(H). The midpoint potential of the [2Fe-2S] protein was determined for the first time at physiological temperature to be +290 mV at pH 8.7. The reduced minus oxidized difference extinction coefficients of the alpha-bands of the cytochromes were calculated as 11.5, 19, and 6.7 mM(-1) cm(-1) for cytochromes c(1), b(H), and b(L), respectively. A novel method has been developed to quantify protonation reactions of the complex during the redox reactions of its cofactors by evaluation of the buffer signals in the midinfrared region. Values will be discussed in relation to the pH dependence of the midpoint potentials.     

Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.     

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.     

Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc(1) complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc(1) complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 degrees C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Angstroms resolution.     

Oxidoreductase activity of chromatophores and purified cytochrome bc 1 complex from Rhodobacter sphaeroides: a possible role of cardiolipin

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60?±?2 to 93?±?3?mol cyt c s(-1) (mol total cyt c)(-1). Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc ( 1 ) complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc ( 1 ) complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc ( 1 ) complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophore and in purified cyt bc ( 1 ) complex.     

Role of specific lysine residues in the reaction of Rhodobacter sphaeroides cytochrome c2 with the cytochrome bc1 complex

The reaction of Rhodobacter sphaeroides cytochrome c2 with the Rb. sphaeroides cytochrome bc1 complex was studied by using singly labeled cytochrome c2 derivatives. Cytochrome c2 was treated with chlorodinitrobenzoic acid to modify lysine amino groups to negatively charged carboxydinitrophenyllysines and separated into eight different fractions by ion-exchange chromatography on a Whatman SE 53 (sulfoxyethyl)cellulose column. Peptide mapping studies indicated that six of these fractions were modified at single lysine amino groups. Each of the derivatives had the same Vmax value as native cytochrome c2 in the steady-state reaction with the Rb. sphaeroides cytochrome bc1 complex. However, the Km values of the cytochrome c2 derivatives modified at lysines 10, 55, 95, 97, 99, and 106 were found to be larger than that of native cytochrome c2 by factors of 6, 2, 3, 32, 13, and 8, respectively. These results indicate that lysines located in the sequence 97-106 on the left side of the heme crevice have the greatest involvement in binding the cytochrome bc1 complex. The involvement of lysine 97 is especially significant because it is located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c.     

The road to the crystal structure of the cytochrome bc 1 complex from the anoxigenic, photosynthetic bacterium Rhodobacter sphaeroides

The advantages of using bacterial systems to study the mechanism and function of cytochrome bc (1) complexes do not extend readily to their structural investigations. High quality crystals of bacterial complexes have been difficult to obtain despite the enzymes' smaller sizes and simpler subunit compositions compared to their mitochondrial counterparts. In the course of the structure determination of the bc (1) complex from R. sphaeroides, we observed that the growth of only low quality crystals correlated with low activity and stability of the purified complex, which was mitigated in part by introducing a double mutations to the enzyme. The S287R(cyt b)/V135S(ISP) mutant shows 40% increase in electron transfer activity and displays a 4.3 degrees C increase in thermal stability over wild-type enzyme. The amino acid histidine was found important in maintaining structural integrity of the bacterial complex, while the respiratory inhibitors such as stigmatellin are required for immobilization of the iron-sulfur protein extrinsic domain. Crystal quality of the R. sphaeroides bc (1) complex can be improved further by the presence of strontium ions yielding crystals that diffracted X-rays to better than 2.3 A resolution. The improved crystal quality can be understood in terms of participation of strontium ions in molecular packing arrangement in crystal.