Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus:: Characterization of the iron-sulfur centers from the dehydrogenases and investigation of the high-potential iron-sulfur protein function by in vitro reconstitution of the respiratory chain

Rhodothermus marinus, a thermohalophilic bacterium, has a unique electron-transfer chain, containing, besides a cbb(3) and a caa(3) terminal oxidases, a novel cytochrome be complex [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1268-1275]. The membrane-bound iron-sulfur centers of this bacterium were studied by electron paramagnetic resonance (EPR) spectroscopy, leading to the identification of its main electron-transfer complexes. The resonances typical for the Rieske-type centers are not detected. Clusters S1 and S3 from succinate dehydrogenase were identified; interestingly, center S3 is shown to be present in two different conformations, with g values at 2.035, 2.009, and 2.001 and at 2.025, 2.002, and 2.000, Upon addition of NADH and dithionite, EPR signals assigned to resonances characteristic of binuclear and tetranuclear clusters develop and are attributed to the iron-sulfur centers' of complexes I and II. A high-potential iron-sulfur protein- (HiPIP-) type center previously detected in the membranes of this bacterium [Pereira et al. (1994) FEES Lett. 352, 327-330] is shown to belong indeed to a canonical HiPIP, This protein was purified and extensively characterized. It is a small water-soluble protein of similar to 10 kDa, containing a single [4Fe-4S](3+/2+) cluster. The reduction potential, determined by EPR redox titrations in intact and detergent-solubilized membranes as well as by cyclic voltammetry in solution, has a pH-independent value of 260 +/- 20 mV, in the range 6-9. In vitro reconstitution of the R, marinus electron-transfer chain shows that the HiPIP plays a fundamental role in the chain, as the electron shuttle between R. marinus cytochrome be complex and the caa(3) terminal oxidase, being thus simultaneously identified a HiPIP reductase and a HiPIP oxidase.