Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

产电微生物基因调控网络的构建和特异性通路分析

研究产电微生物胞外电子传递过程和机制,发现与产电效率相关的关键基因、通路和代谢物,是微生物燃料电池研究中的关键技术。为了发现在胞外电子传递过程中起到关键作用的基因以及通路,首先利用比较基因组学的方法,以模式微生物大肠杆菌和同属希瓦氏菌的其他菌株为参考,构建了Shewanella.onedensis MR-1的全基因组基因转录调控网络,大大扩展了目前已知的基因调控关系。然后以此网络为基础,结合基于蛋白质相互作用分析得到的胞外电子传递通路,构建了与胞外电子传递直接传递密切相关的细胞色素C编码基因及其相关调控基因构成的子网络,结合全基因组基因表达数据,研究了特异性条件下胞外电子传递的可能通路和基因调控过程。     

A quick solution structure determination of the fully oxidized double mutant K9-10A cytochrome c7 from Desulfuromonas acetoxidans and mechanistic implications

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c₇, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c₇, which is paramagnetic with three paramagnetic centers, has been determined via ¹H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 ± 0.17 Å and 1.05 ± 0.14 Å for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short -sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 ± 0.07 Å and 0.92 ± 0.06 Å for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c₇ with [Fe] hydrogenase, inducing a change in K _m. This finding is in agreement with the identification of the protein area around heme IV as the interacting site.     

Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing

Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).     

Lithoautotrophic Growth of the Freshwater Colorless Sulfur Bacterium Beggiatoa “leptomitiformis”D-402

The freshwater colorless sulfur bacterium Beggiatoa leptomitiformisD-402 was shown to be capable of lithoautotrophic growth in a batch culture under microoxic conditions at O₂concentrations in the medium of no higher than 0.5 mg/l. The cell yield was maximum at a dissolved oxygen concentration of 0.15 mg/l. A high activity level of key enzymes of the Calvin cycle and of enzymes involved in dissimilatory oxidation of thiosulfate was recorded in the cells. The high rate of CO₂assimilation (112–139 nmol/(min mg protein)) and the cell yield (12 mg dry cells/mmol thiosulfate oxidized), 91–92% of which was accounted for by CO₂carbon, were close to those typical of autotrophic bacteria. Thiosulfate was oxidized almost completely to sulfate, and the fraction of intracellular sulfur in the final products did not exceed 0.2–1.7% of the thiosulfate sulfur. The cell membrane fraction contained cytochromes (b + o) and two cytochromes cwith M _rof 23 and 26 kDa; the soluble fraction contained cytochrome cwith M _rof 12 kDa.     

Three-dimensional model of the human aromatase enzyme and density functional parameterization of the iron-containing protoporphyrin IX for a molecular dynamics study of heme-cysteinato cytochromes

Mammalian cytochromes P450 (CYP) are enzymes of great biological and pharmaco-toxicological relevance. Due to their membrane-bound nature, the structural characterization of these proteins is extremely difficult, and therefore computational techniques, such as comparative modeling, may help obtaining reliable structures of members of this family. An important feature of CYP is the presence of an iron-containing porphyrin group at the enzyme active site. This calls for quantum chemical calculations to derive charges and parameters suitable for classical force field-based investigations of this proteins family. In this report, we first carried out density functional theory (DFT) computations to derive suitable charges for the Fe2+-containing heme group of P450 enzymes. Then, by means of the homology modeling technique, and taking advantage of the recently published crystal structure of the human CYP2C9, we built a new model of the human aromatase (CYP19) enzyme. Furthermore, to study the thermal stability of the new model as well as to test the suitability of the new DFT-based heme parameters, molecular dynamics (MD) simulations were carried out on both CYP2C9 and CYP19. Finally, the last few ns of aromatase MD trajectories were investigated following the essential dynamics protocol that allowed the detection of some correlated motions among some protein domains.     

Respiratory Chain Components of Leishmania tropica Promastigotes*

Synopsis. Crude preparations of kinetoplast vesicles were used to investigate the respiratory chain components in Leishmania tropica promastigotes. In difference spectra from enzymically and chemically reduced preparations, cytochrome b was the predominant component. By utilizing special assays designed to minimize the influence of cytochrome b on difference spectra, cytochromes a, a₃, and c₃₃₃ were demonstrated. Difference spectra from chemically reduced preparations indicated that pyridine nucleotides (NADH) and flavoproteins were also part of the respiratory chain. The presence of these components as well as their response to respiratory inhibitors and ascorbate provide evidence for the presence of a typical trypanosomatid respiratory chain in L. tropica promastigotes.     

Mechanisms of energy dissipation in peanut under water stress

Effect of drought on the mechanisms of energy dissipation was evaluated in two-month-old Arachis hypogaea cvs. 57–422, 73–30, and GC 8–35. Plants were submitted to three treatments: control (C), mild water stress (S1), and severe water stress (S2). Photosynthetic performance was evaluated as the Hill and Mehler reactions. These activities were correlated with the contents of the low and high potential forms of cytochrome (cyt) b ₅₅₉, plastoquinone, cyt b ₅₆₃, and cyt f. Additionally, the patterns of carotenoids and chlorophylls (Chls), as well as the alterations of Chl a fluorescence parameters were studied. Under mild water stress the regulatory mechanism at the antennae level was effective for 57–422 and GC 8–35, while in the cv. 73–30 an overcharge of photosynthetic apparatus occurred. Relative to this cv., under S1 the stability of carotene and the dissipative cycle around photosystem (PS) 2 became an important factor for the effective protection of the PS2 reaction centres. The cyclic electron flow around PS1 was important for energy dissipation under S1 only for the cvs. 57–422 and 73–30.     

Distribution of cytochromes in methanogenic bacteria

Abstract Various methanogenic bacteria belonging to the orders Methanobacteriales, Methanococcales , and Methanomicrobiales were examined for the presence of cytochromes. Those methanogens which are capable of growing only on H ₂+ CO ₂ or formate were found to lack cytochromes. However, membrane-bound cytochromes were detected in species able to utilize methanol, methylamines or acetate.