RESUMEN

Most organisms contain a single Rieske/cytb complex. This enzyme can be integrated in any respiratory or photosynthetic electron transfer chain that is quinone-based and sufficiently energy rich to allow for the turnover of three enzymes - a quinol reductase, a Rieske/cytb complex and a terminal oxidase. Despite this universal usability of the enzyme a variety of phylogenetically distant organisms have multiple copies thereof and no reason for this redundancy is obvious. In this review we present an overview of the distribution of multiple copies among species and describe their properties from the scarce experimental results, analysis of their amino acid sequences and genomic context. We discuss the predicted redox properties of the Rieske cluster in relation to the nature of the pool quinone. It appears that acidophilic iron-oxidizing bacteria specialized one of their two copies for reverse electron transfer, archaeal Thermoprotei adapted their three copies to the interaction with different oxidases and several, phylogenetically unrelated species imported a second complex with a putative heme ci that may confer some yet to be determined properties to the complex. These hypothesis and all the more the so far completely unexplained cases call for further studies and we put forward a number of suggestions for future research that we hope to be stimulating for the field. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Asunto(s)

Proteínas Arqueales/genética , Proteínas Bacterianas/genética , Citocromos b/genética , Complejo III de Transporte de Electrones/genética , Archaea/clasificación , Archaea/genética , Archaea/metabolismo , Proteínas Arqueales/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Benzoquinonas/metabolismo , Citocromos b/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Oxidación-Reducción , Filogenia , Especificidad de la Especie

RESUMEN

Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) argued that the bacterial strain GFAJ-1 can vary the elemental composition of its biomolecules by substituting arsenic for phosphorus. Although their data show that GFAJ-1 is an extraordinary extremophile, consideration of arsenate redox chemistry undermines the suggestion that arsenate can replace the physiologic functions of phosphate.

Asunto(s)

Arsénico/metabolismo , Halomonadaceae/metabolismo , Fósforo/metabolismo , Adaptación Fisiológica , Arseniatos/química , Arsénico/análisis , Arsénico/química , Arsenitos/química , Halomonadaceae/crecimiento & desarrollo , Estructura Molecular , Oxidación-Reducción , Fosfatos/química

RESUMEN

More than a decade ago, Heliobacteria were recognised to contain a Rieske/cytb complex in which the cytochrome b subunit is split into two separate proteins, a peculiar feature characteristic of the cyanobacterial and plastidic b (6) f complex. The common presence of RCI-type reaction centres further emphasise possible evolutionary links between Heliobacteria, Chlorobiaceae and Cyanobacteria. In this contribution, we further explore the evolutionary relationships among these three phototrophic lineages by both molecular phylogeny and consideration of phylogenetic marker traits of the superfamily of Rieske/cytb complexes. The combination of these two methods suggests the existence of a "green" clade involving many non-phototrophs in addition to the mentioned RCI-type photosynthetic organisms. Structural and functional idiosyncrasies are (re-)interpreted in the framework of evolutionary biology and more specifically evolutionary bioenergetics.

Asunto(s)

Citocromos b/genética , Complejo III de Transporte de Electrones/genética , Bacterias Grampositivas/enzimología , Filogenia , Citocromos b/química , Complejo III de Transporte de Electrones/química , Evolución Molecular

RESUMEN

Data on structure and function of the Rieske/cytb complex from Heliobacteria are scarce. They indicate that the complex is related to the b (6) f complex in agreement with the phylogenetic position of the organism. It is composed of a diheme cytochrome c, and a Rieske iron-sulfur protein, together with transmembrane cytochrome b (6) and subunit IV. Additional small subunits may be part of the complex. The cofactor content comprises heme c (i), first discovered in the Q(i) binding pocket of b (6) f complexes. The redox midpoint potentials are more negative than in b (6) f complex in agreement with the lower redox midpoint potentials (by about 150 mV) of its reaction partners, menaquinone, and cytochrome c (553). The enzyme is implicated in cyclic electron transfer around the RCI. Functional studies are favored by the absence of antennae and the simple photosynthetic reaction chain but are hampered by the high oxygen sensitivity of the organism, its chlorophyll, and lipids.

Asunto(s)

Grupo Citocromo b/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Bacterias Grampositivas/metabolismo , Secuencia de Aminoácidos , Carotenoides/metabolismo , Clorofila/metabolismo , Coenzimas/metabolismo , Grupo Citocromo b/química , Complejo III de Transporte de Electrones/química , Metabolismo Energético , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo

RESUMEN

Kulp et al. (Reports, 15 August 2008, p. 967) described a bacterium able to photosynthetically oxidize arsenite [As(III)] via arsenate [As(V)] reductase functioning in reverse. Based on their phylogenetic analysis of As(V) reductase, they proposed that this enzyme was responsible for the anaerobic oxidation of As(III) in the Archean. We challenge this proposition based on paleogeochemical, bioenergetic, and phylogenetic arguments.

Asunto(s)

Arseniato Reductasas/metabolismo , Arsenitos/metabolismo , Bacterias/metabolismo , Biopelículas , Manantiales de Aguas Termales/microbiología , Fotosíntesis , California , Oxidación-Reducción , Filogenia

RESUMEN

The reacton centre I (RCI)-type photosystems from plants, cyano-, helio- and green sulphur bacteria are compared and the essential properties of an archetypal RCI are deduced. Species containing RCI-type photosystems most probably cluster together on a common branch of the phylogenetic tree. The predicted branching order is green sulphur, helio- and cyanobacteria. Striking similarities between RCI- and RCII-type photosystems recently became apparent in the three-dimensional structures of photosystem I (PSI), PSII and RCII. The phylogenetic relationship between all presently known photosystems is analysed suggesting (a) RCI as the ancestral photosystem and (b) the descendence of PSII from RCI via gene duplication and gene splitting. An evolutionary model trying to rationalise available data is presented.

Asunto(s)

Proteínas del Complejo del Centro de Reacción Fotosintética/química , Secuencia de Aminoácidos , Bacterias/química , Chlorobi/química , Cianobacterias/química , Metabolismo Energético , Evolución Molecular , Datos de Secuencia Molecular , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema I , Filogenia , Estructura Secundaria de Proteína , Alineación de Secuencia

RESUMEN

Two distinct class I (monoheme) c-type cytochromes from the hyperthermophilic bacterium Aquifex aeolicus were studied by biochemical and biophysical methods (i.e., optical and EPR spectroscopy, electrochemistry). The sequences of these two heme proteins (encoded by the cycB1 and cycB2 genes) are close to identical (85% identity in the common part of the protein) apart from the presence of an N-terminal stretch of 62 amino acid residues present only in the cycB1 gene. A soluble cytochrome was purified and identified by N-terminal sequencing as the cycB2 gene product. It showed an alpha-peak at 555 nm, an E(m) value of +220 mV, and electron paramagnetic resonance parameters of gz = 2.89, gy = 2.287, and gx = 1.52. A firmly membrane-bound cytochrome characterized by nearly identical properties was detected and attributed to the cycB1 gene product. The very high degree of homology of its N-terminal part to cytochrome c553 from Heliobacterium gestii strongly suggests it to be anchored to the membrane via N-terminally attached lipid molecules. The two heme proteins were named cytochrome c555s (soluble) and cytochrome c555m (membranous). Electron paramagnetic resonance on partially ordered membrane multilayers suggests that the solvent-exposed heme domain of cytochrome c555m is flexible with respect to the membrane plane. Possible functional roles for both cytochromes are discussed.

Asunto(s)

Bacterias/enzimología , Proteínas Bacterianas , Citocromos c , Citocromos/genética , Adaptación Biológica , Secuencia de Aminoácidos , Citocromos/química , Citocromos/fisiología , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Oxidación-Reducción , Filogenia , Homología de Secuencia de Aminoácido , Temperatura

RESUMEN

The orientation of the membrane-attached cytochrome b(558/566)-haem with respect to the membrane was determined by electron paramagnetic resonance spectroscopy on two-dimensionally ordered oxidised membrane fragments from Sulfolobus acidocaldarius. Unlike the other redox centres in the membrane, the cytochrome b(558/566)-haem was found to cover a range of orientations between 25 degrees and 90 degrees. The described results are reminiscent of those obtained on the Rieske cluster of bc complexes and indicate that the membrane-extrinsic domain of cytochrome b(558/566) can perform pivoting motion between two extreme positions. Such a conformational flexibility is likely to play a role in electron transfer with its redox partners.

Asunto(s)

Grupo Citocromo b/química , NADPH Oxidasas , Sulfolobus acidocaldarius/química , Secuencia de Aminoácidos , Membrana Celular/química , Grupo Citocromo b/genética , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Sulfolobus/química , Sulfolobus/genética , Sulfolobus acidocaldarius/genética

RESUMEN

Primary structures, functional characteristics and phylogenetic relationships of subunits of cytochrome bc complexes from phylogenetically diverse bacterial and archaeal species were analysed. A single case of lateral gene transfer, i.e. the import of an epsilon-proteobacterial cytochrome bc(1) complex into Aquificales, was identified. For the enzyme in the remainder of the species studied, the obtained phylogenies were globally in line with small subunit rRNA trees. The distribution of a few key phylogenetic markers, such as contiguousness of cytochrome b, nature of the c-type subunit or spacing between b-heme ligands, are discussed. A localised modification of previous tree topologies is proposed on the basis of the obtained data. The comparison of extant enzymes furthermore allowed us to define the minimal functional and evolutionary core of the enzyme. The data furthermore suggest that the ancestral enzyme was put together from subunits that previously had played a role in other electron transfer chains.

Asunto(s)

Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/genética , Evolución Molecular , Filogenia , Secuencia de Aminoácidos , Archaea/enzimología , Chlorobi/enzimología , Cianobacterias/enzimología , Complejo III de Transporte de Electrones/metabolismo , Bacterias Grampositivas/enzimología , Datos de Secuencia Molecular , Proteobacteria/enzimología , Recombinación Genética , Alineación de Secuencia

RESUMEN

A new soluble c-type cytochrome has been purified to homogeneity from the acidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characterized by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (as determined by mass spectroscopy) and a pI value of 8. Optical redox titrations at pH 4.0 revealed the presence of two distinguishable redox species with an E(m) of 510 mV and an E(m) of 430 +/- 20 mV. EPR spectra recorded for this heme protein demonstrated the presence of stoichiometric amounts of two low-spin hemes with a g(z)() of 3.08 (510 mV species) and a g(z)() of 3.22 (430 mV species). Modifications of the physicochemical properties of the cytochrome were observed on complex formation with the blue copper protein rusticyanin, another soluble electron carrier in the genus Thiobacillus. N-Terminal sequencing yielded the polypeptide sequence up to the 50th residue. The determined sequence was found to be present (at 100% amino acid identity) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresponding full-length protein turned out to be surprisingly similar (34.5% amino acid identity) to another c(4)-type diheme protein from T. ferrooxidans BRGM [Cavazza, C., et al. (1996) Eur. J. Biochem. 242, 308-314], the gene of which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteristics of both c(4) cytochromes present in the same bacteria are compared, and the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.

Asunto(s)

Grupo Citocromo c/química , Thiobacillus/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Azurina/análogos & derivados , Azurina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Espectrofotometría

RESUMEN

The g-tensor orientation of the chemically reduced Rieske cluster in cytochrome bc(1) complex from Rhodovulum sulfidophilum with respect to the membrane was determined in the presence and absence of inhibitors and in the presence of oxidized and reduced quinone in the quinol-oxidizing-site (Q(o)-site) by EPR on two-dimensionally ordered samples. Almost identical orientations were observed when oxidized or reduced quinone, stigmatellin, or 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole was present. Occupancy of the Q(o)-site by myxothiazole induced appearance of a minority population with a substantially differing conformation and presence of E-beta-methoxyacrylate-stilbene significantly reduced the contribution of the major conformation observed in the other cases. Furthermore, when the oxidized iron-sulfur cluster was reduced at cryogenic temperatures by the products of radiolysis, the orientation of its magnetic axes was found to differ significantly from that of the chemically reduced center. The "irradiation-induced" conformation converts to that of the chemically reduced center after thawing of the sample. These results confirm the effects of Q(o)-site inhibitors on the equilibrium conformation of the Rieske iron-sulfur protein and provide evidence for a reversible redox-influenced interconversion between conformational states. Moreover, the data obtained with the iron-sulfur protein demonstrate that the conformation of "EPR-inaccessible" reduction states of redox centers can be studied by inducing changes of redox state at cryogenic temperatures. This technique appears applicable to a wide range of comparable electron transfer systems performing redox-induced conformational changes.

Asunto(s)

Proteínas Hierro-Azufre/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón/métodos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Rayos gamma , Oxidación-Reducción , Fotólisis , Polienos , Conformación Proteica , Rhodobacter , Estilbenos , Temperatura

RESUMEN

The blue copper protein rusticyanin isolated from the acidophilic proteobacterium Thiobacillus ferrooxidans displays a pH-dependent redox midpoint potential with a pK value of 7 on the oxidized form of the protein. The nature of the alterations of optical and EPR spectra observed above the pK value indicated that the redox-linked deprotonation occurs on the epsilon-nitrogen of the histidine ligands to the copper ion. Complex formation between rusticyanin and its probable electron transfer partner, cytochrome c(4), induced a decrease of rusticyanin's redox midpoint potential by more than 100 mV together with spectral changes similar to those observed above the pK value of the free form. Complex formation thus substantially modifies the pK value of the surface-exposed histidine ligand to the copper ion and thereby tunes the redox midpoint potential of the copper site. Comparisons with reports on other blue copper proteins suggest that the surface-exposed histidine ligand is employed as a redox tuning device by many members of this group of soluble electron carriers.

Asunto(s)

Acidithiobacillus thiooxidans/metabolismo , Azurina/análogos & derivados , Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Azurina/química , Azurina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cobre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica

RESUMEN

We have altered the N terminus of cytochrome f by site-directed mutagenesis of the chloroplast petA gene in Chlamydomonas reinhardtii. We have replaced the tyrosine residue, Tyr(32), located immediately downstream of the processing site Ala(29)-Gln(30)-Ala(31) by a proline. Tyr(32) is the N terminus of the mature protein and serves as the sixth axial ligand to the heme iron. This mutant, F32P, accumulated different forms of holocytochrome f and assembled them into the cytochrome b(6)f complex. The strain was able to grow phototrophically. Our results therefore contradict a previous report (Zhou, J., Fernandez-Velasco, J. G., and Malkin, R. (1996) J. Biol. Chem. 271, 1-8) that a mutation, considered to be identical to the mutation described here, prevented cytochrome b(6)f assembly. A comparative functional characterization of F32P with F29L-31L, a site-directed processing mutant in which we had replaced the processing site by a Leu(29)-Gln(30)-Leu(31) sequence (2), revealed that both mutants accumulate high spin cytochrome f, with an unusual orientation of the heme and low spin cytochrome f with an alpha-band peak at 552 nm. Both hemes have significantly lower redox potentials than wild type cytochrome f. We attribute the high spin form to uncleaved pre-holocytochrome f and the low spin form to misprocessed forms of cytochrome f that were cleaved at a position different from the regular Ala(29)-Gln-Ala(31) motif. In contrast to F29L-31L, F32P displayed a small population of functional cytochrome f, presumably cleaved at Ala(29), with characteristics close to those of wild type cytochrome f. The latter form would account for cytochrome b(6)f turnover and photosynthetic electron transfer that sustain phototrophic growth of F32P.

Asunto(s)

Chlamydomonas reinhardtii/enzimología , Citocromos/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chlamydomonas reinhardtii/genética , Citocromos/química , Citocromos f , Cartilla de ADN , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Precursores Enzimáticos/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Espectrometría de Fluorescencia

RESUMEN

Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from Desulfuromonas acetoxidans. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium D. acetoxidans, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of D. acetoxidans, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium.

Asunto(s)

Hidrogenasas/metabolismo , Bacterias Reductoras del Azufre/enzimología , Secuencia de Aminoácidos , Grupo Citocromo c/metabolismo , Bacterias Anaerobias Gramnegativas/enzimología , Hidrogenasas/aislamiento & purificación , Datos de Secuencia Molecular

RESUMEN

The interaction of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with the Rieske protein of the chloroplast b6f complex has been studied by EPR. All three redox states of DBMIB were found to interact with the iron-sulphur cluster. The presence of the oxidised form of DBMIB altered the equilibrium distribution of the Rieske protein's conformational substates, strongly favouring the proximal position close to heme bL. In addition to this conformational effect, DBMIB shifted the pK-value of the redox-linked proton involved in the iron-sulphur cluster's redox transition by about 1.5 pH units towards more acidic values. The implications of these results with respect to the interaction of the native quinone substrate and the Rieske cluster in cytochrome bc complexes are discussed.

Asunto(s)

Cloroplastos/metabolismo , Grupo Citocromo b/metabolismo , Dibromotimoquinona/metabolismo , Complejo III de Transporte de Electrones , Proteínas Hierro-Azufre/metabolismo , Ácido Ascórbico/metabolismo , Sitios de Unión , Grupo Citocromo b/antagonistas & inhibidores , Complejo de Citocromo b6f , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/antagonistas & inhibidores , Proteínas Hierro-Azufre/química , Oxidación-Reducción , Protones

RESUMEN

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.

Asunto(s)

Chlamydomonas reinhardtii/enzimología , Grupo Citocromo b/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Chlamydomonas reinhardtii/genética , Cloroplastos/enzimología , Cloroplastos/metabolismo , Secuencia Conservada/genética , Grupo Citocromo b/química , Grupo Citocromo b/genética , Complejo de Citocromo b6f , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Activación Enzimática , Fluorescencia , Cinética , Complejos de Proteína Captadores de Luz , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutación , Oxidación-Reducción , Péptidos/metabolismo , Fosforilación , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema I , Plastoquinona/análogos & derivados , Plastoquinona/metabolismo , Temperatura

RESUMEN

The redox components of the cytochrome bc1 complex from the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans were investigated by potentiometric and spectroscopic techniques. Optical redox titrations demonstrated the presence of two b-type hemes with differing redox midpoint potentials at pH 7.4 (-169 and + 20 mV for bL and bH, respectively). At pH 3.5, by contrast, both hemes appeared to titrate at about +20 mV. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide, and stigmatellin induced distinguishable shifts of the b hemes' alpha-bands, providing evidence for the binding of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide near heme bH (located on the cytosolic side of the membrane) and of stigmatellin near heme bL (located on the periplasmic side of the membrane). The inhibitors stigmatellin, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone affected the EPR spectrum of the Rieske iron-sulfur center in a way that differs from what has been observed for cytochrome bc1 or b6f complexes. The results obtained demonstrate that the T. ferrooxidans complex, although showing most of the features characteristic for bc1 complexes, contains unique properties that are most probably related to the chemolithotrophicity and/or acidophilicity of its parent organism. A speculative model for reverse electron transfer through the T. ferrooxidans complex is proposed.

Asunto(s)

Complejo III de Transporte de Electrones/química , Proteínas Hierro-Azufre/química , Thiobacillus/enzimología , Antimicina A/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/metabolismo , Compuestos Ferrosos/metabolismo , Hemo/análogos & derivados , Hemo/química , Concentración de Iones de Hidrógeno , Hidroxiquinolinas/farmacología , Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Metacrilatos , Modelos Químicos , Oxidación-Reducción , Polienos/farmacología , Potenciometría , Fuerza Protón-Motriz/efectos de los fármacos , Especificidad de la Especie , Espectrofotometría , Termodinámica , Tiazoles/farmacología

RESUMEN

The Rieske proteins of two phylogenetically distant acidophilic organisms, i.e. the proteobacterium Thiobacillus ferrooxidans and the crenarchaeon Sulfolobus acidocaldarius, were studied by EPR. Redox titrations at a range of pH values showed that the Rieske centers of both organisms are characterized by redox midpoint potential-versus-pH curves featuring a common pK value of 6.2. This pK value is significantly more acidic (by almost 2 pH units) than that of Rieske proteins in neutrophilic species. The orientations of the Rieske center's g tensors with respect to the plane of the membrane were studied between pH 4 and 8 using partially ordered samples. At pH 4, the Sulfolobus Rieske cluster was found in the "typical" orientation of chemically reduced Rieske centers, whereas this orientation changed significantly on going toward high pH values. The Thiobacillus protein, by contrast, appeared to be in the "standard" orientation at both low and high pH values. The results are discussed with respect to the molecular parameters conveying acid resistance and in light of the recently demonstrated long-range conformational movement of the Rieske protein during enzyme turnover in cytochrome bc1 complexes.

Asunto(s)

Complejo III de Transporte de Electrones/química , Proteínas Hierro-Azufre/química , Sulfolobus acidocaldarius/enzimología , Thiobacillus/enzimología , Grupo Citocromo b , Grupo Citocromo c , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Potenciometría

RESUMEN

A highly active, large-scale preparation of cytochrome bc1 complex has been obtained from the photosynthetic purple bacterium Rhodovulum (Rhv.) sulfidophilum. It has been characterized using mass spectrometry, quinone and lipid analysis as well as inhibitor binding. About 35 mg of pure complex can be obtained from 1 g of membrane protein. EPR spectroscopy and optical titrations have been used to obtain the redox midpoint potentials of the cofactors. The Em-value of 310 mV for the Rieske protein is the most positive midpoint potential for this protein in a bc1 complex so far. The bc1 complex from Rhv. sulfidophilum is very stable and consists of three subunits and a 6-kDa polypeptide. The complex appears as a dimer in solution and contains six quinone molecules per monomer which are tightly bound. EPR spectroscopy shows that the Q(o) site is highly occupied. High detergent concentrations convert the complex into an inactive, monomeric form that has lost the Rieske protein as well as the quinones and the 6-kDa component.

Asunto(s)

Proteínas Bacterianas/química , Complejo III de Transporte de Electrones/química , Quinonas/química , Rhodobacter/enzimología , Secuencia de Aminoácidos , Detergentes/farmacología , Dimerización , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Estabilidad de Enzimas , Proteínas Hierro-Azufre/química , Lípidos/análisis , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría Ultravioleta

RESUMEN

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (Em) of +160 mV in the pH range of 6 to 7.7 and a decreasing Em (-60 to -80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its Em-versus-pH curve, in green sulfur bacteria are discussed.

Asunto(s)

Chlorobi/química , Grupo Citocromo b/química , Grupo Citocromo c/química , Complejo III de Transporte de Electrones , Proteínas Hierro-Azufre/química , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética