Cooperative interaction of high-potential hemes in the cytochrome subunit of the photosynthetic reaction center of bacterium <Emphasis Type="Italic">Ectothiorhodospira shaposhnikovii</Emphasis>

Cooperative interaction of the high-potential hemes (C_h) in the cytochrome subunit of the photosynthesizing bacterium Ectothiorhodospira shaposhnikovii was studied by comparing redox titration curves of the hemes under the conditions of pulse photoactivation inducing single turnover of electron-transport chain and steady-state photoactivation, as well as by analysis of the kinetics of laser-induced oxidation of cytochromes by reaction center (RC). A mathematical model of the processes of electron transfer in cytochrome-containing RC was considered. Theoretical analysis revealed that the reduction of one heme C_h facilitated the reduction of the other heme, which was equivalent to a 60 mV positive shift of the midpoint potential. In addition, reduction of the second heme C_h caused a three-to four-fold acceleration of the electron transfer from the cytochrome subunit to RC. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1540–1547.     

Redox chemistry of low-pH forms of tetrahemic cytochrome c3

Desulfovibrio vulgaris Hildenborough cytochrome c₃ contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c₃ can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV–visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.     

Polymorphisms of four hepatic cytochromes P-450 in twenty-eight inbred strains of rat

Two-dimensional gel electrophoresis of hepatic microsomes from phenobarbital-treated animals was used to analyze electrophoretic/regulatory polymorphisms for cytochromes P-450b, P-450e, P-450g, and P-450h in 28 inbred strains of rat. Previous studies with outbred rats revealed the existence of four electrophoretic variants for P-450b, two for P-450e, and three for P-450h as well as two regulatory alleles for P-450g. With the exception of one allozymic form of P-450h, all of these alleles as well as a novel (null) allele for P-450e were found to be homozygous in at least two of the inbred strains tested. Eight phenotypes for combinations of these four cytochromes P-450 were observed. Inbred strains were identified that can be used in studies on the structure/function of unique cytochrome P-450-allozymes and in genetic crosses to map the four distinct cytochrome P-450 genes.This work was supported by National Institutes of Health Biomedical Research Support Grant SO7RR07208.     

On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes

Four distinct integration/translocation routes into/across thylakoid membranes have recently been deduced for nuclear-encoded polypeptides of the photosynthetic membrane. Corresponding information for the plastid-encoded protein complement is lacking. We have investigated this aspect with in-organello assays employing chimeric constructs generated with codon-correct cassettes for genes of plastid-encoded thylakoid proteins, and appropriate transit peptides from six nuclear genes, representing three targeting classes, as a strategy. The three major plastid-encoded components of the cytochrome b ₆  f complex, namely pre-apocytochrome f, (including apocytochrome f, and pre-apocytochrome f lacking the C-terminal transmembrane segment), cytochrome b ₆ , and subunit IV, which differ in the number of their transmembrane segments, were studied. Import into chloroplasts could be observed in all instances but with relatively low efficiency. Thylakoid integration can occurr post-translationally, but only components with secretory/secretory pathway (SEC)-route-specific epitopes were correctly assembled with the cytochrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b ₆ and subunit IV operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably, contribute to this process. The N-termini of cytochrome b ₆ and subunit IV appear to reside on the same (lumenal) side of the membrane, consistent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed. Received: 18 September 1996 / Accepted: 15 October 1996     

Steady-State Proton Translocation in Bovine Heart Mitochondrial bc 1 Complex Reconstituted into Liposomes

The effect of different anions on the steady-state proton translocation in bovine bc ₁ complex reconstituted in liposomes was studied. The H⁺/e ^– ratio for vectorial proton translocation is at the steady state definitely lower than that measured at level flow, (0.3 vs. 1.0). The presence of azide or arachidonate at micro- and submicromolar concentrations, respectively, gave a substantial reactivation of the proton pumping activity at the steady state, without any appreciable effect on respiration-dependent transmembrane pH difference. Addition of azide to turning-over bc ₁ vesicles also caused a transition of b cytochromes toward oxidation. The results are discussed in terms of possible involvement of an acidic residue in the protonation of the semiquinone/quinol couple at the N side of the membrane.     

Structure of the three-haem core of cytochrome c 551.5 determined by 1H NMR

 The trihaem cytochrome c _551.5, formerly known as cytochrome c ₇, from the organism Desulfuromonas acetoxidans, has been studied in the reduced state by 2D proton NMR. The haem proton resonances were assigned, and several nuclear Overhauser enhancements (NOEs) between resonances arising from different haems were detected and assigned. The relative orientations of the three haems were calculated by fitting both the intensities of the interhaem NOEs and the magnitudes of the ring current shifts of the haem resonances, following the strategy previously used by the authors to reassess the X-ray structure of the haem core in tetrahaem cytochrome c ₃ from Desulfumicrobium baculatum. It is concluded that, although the comparison of the protein sequence with those of the tetrahaem cytochromes c ₃ shows that in cytochrome c _551.5 about 40% of the sequence is deleted, including the region involved in the attachment of the second of the four haems, this does not induce any significant rearrangement of the remaining three haems other than a slight decrease in the iron-iron distance between two of the haems, namely those corresponding to haems I and IV of cytochrome c ₃. Received: 2 February 1996 / Accepted: 26 March 1996     

生物地球化学锰循环中的微生物胞外电子传递机制

微生物是生物地球化学元素循环的重要驱动者,在锰等变价金属元素的氧化还原过程中起着至关重要的作用。近年来,Mn(Ⅲ)的发现以及在一些环境中的广泛存在,丰富了人们对Mn(Ⅲ)以及自然界锰循环过程的认识。研究发现,锰的生物地球化学循环,尤其是锰还原过程,与微生物胞外电子传递紧密相关,且目前已知的5种胞外电子传递机制均与锰还原有关联。因此,本文综述了锰的生物地球化学循环及其意义,并从微生物胞外电子传递的机制、微生物介导锰氧化、微生物介导锰还原等3个方面来介绍参与锰循环的微生物多样性;以及微生物地球化学锰循环的环境意义。对微生物参与锰循环过程的研究不仅可以进一步丰富相关理论,同时也能推动生物除锰、污染物原位修复及生物冶金等应用领域的发展。     

7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching,suggesting binding at multiple protein sites

Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern–Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K_d 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K_d 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K_d 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K_d 0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.