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Rhodothermus marinus, a thermohalophilic Gram negative bacterium, contains a type I NADH/ quinone oxidoreductase (complex I). Its purification was optimized, yielding large amounts of pure and active protein. Furthermore, the stoichiometry of NADH oxidation and quinone reduction was shown to be 1:1. The large amounts of protein enabled a thorough characterization by electron paramagnetic resonance (EPR) spectroscopy at different temperatures and microwave powers, using NADH, NADPH, and dithionite as reducing agents. A minimum of two [2Fe-2S]²+/¹+ and four [4Fe-4S]²+/¹+ centers were observed in the purified complex. Redox titrations monitored by EPR spectroscopy made possible the determination of the reduction potentials of the iron–sulfur centers; with the exception of one of the [4Fe-4S]²+/¹+ centers, which has a lower reduction potential, all the other centers have reduction potentials of -240 ± 20 mV, pH 7.5. [ABSTRACT FROM AUTHOR]

A sulfide:quinone oxidoreductase (SQR) was isolated from the membranes of the hyperthermoa-cidophilic archaeon Acidianus ambivalens, and its X-ray structure, the first reported for an SQR, was determined to 2.6 A resolution. This enzyme was functionally and structurally characterized and was shown to have two redox active sites: a covalently bound FAD and an adjacent pair of cysteine residues. Most interestingly, the X-ray structure revealed the presence, of a chain of three sulfur atoms bridging those two cysteine residues; The possible implications of this observation in the catalytic mechanism for sulfide oxidation are discussed, and the role of SQR in the sulfur dependent bioenergetics of A. ambivalens, linked to oxygen reduction, is addressed. [ABSTRACT FROM AUTHOR]

A comprehensive study of the thermodynamic redox behavior of the hemes from the cbb3 oxygen reductase from Bradyrhizobium japonicum was performed. This enzyme is a member of the C-type heme-copper oxygen reductase superfamily and has three subunits with six redox centers: four low-spin hemes and a high-spin heme and one copper ion, composing the site where oxygen is reduced. In this analysis, the visible spectra and redox properties of the five heme centers were deconvoluted. Their redox profiles and the pH dependence of the midpoint reduction potentials (redox-Bohr effect) were investigated. The reference reduction potentials (defined for a state where all centers are reduced) and homotropic interaction potentials were determined in the framework of a model of pairwise interacting redox centers. At pH 7.7, the reference reduction potentials for the three hemes c are 390, 300, and 220 mY, with low interaction potentials between them, weaker than -15 mV. For hemes b and b3, reference reduction potentials of 375 and 290 mV, respectively, were obtained; these two redox centers show an interaction potential weaker than -60 mV. The midpoint reduction potentials of all five hemes are pH-dependent. The study of these thermodynamic parameters is important in understanding the coupling mechanism of the redox and chemical processes during oxygen reduction. The analysis of the thermodynamic redox behavior of the cbb3 oxygen reductase contributes to the investigation of the mechanism of electron transfer and proton translocation by heme-copper oxygen reductases in general and indicates a thermodynamic coupling for the electron and proton transfer mechanisms. [ABSTRACT FROM AUTHOR]