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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]

The thermal unfolding of the plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium, is herein described. The main objective of this work is to identify structural factors responsible for the higher stability observed in proteins from thermophilic organisms. With the aid of fluorescence spectroscopy, EPR, and NMR, the factors influencing the unfolding process of the protein were investigated, and procedures for its study have been standardized. The different spectroscopic techniques used provided consistent results showing that the thermal unfolding of plastocyanin is irreversible under all the conditions investigated and that this irreversibility does not appear to be related to the presence of oxygen. The oxidized plastocyanin species has proven to be more stable than the reduced one, with respect to both the required temperature for protein unfolding (up to a 9 °C difference between the two forms) and the kinetics of the process. The behavior of this plastocyanin contrasts with that of other cupredoxins whose unfolding had previously been studied. The unfolding pH dependence and kinetic studies indicate a process with a tight control around the physiological pH in which plastocyanin plays its redox role and the protein's isoelectric point (5.2), suggesting a close compromise between function and stability. [ABSTRACT FROM AUTHOR]

The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivaleris (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 ± 20 mV for the former and 390 ± 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CUB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO. [ABSTRACT FROM AUTHOR]