Sodium-dependent steps in the redox reactions of the Na+-motive NADH : quinone oxidoreductase from Vibrio harveyi

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR s ignals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E-m, of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme w as approximately the same under a variety of redox conditions. The time cou rse of Na+-NQR reduction by NADH indicated the presence of at least two dif ferent flavin species. Reduction of the first species (most likely, a FAD n ear the NADH dehydrogenase site) was very rapid in both the presence and ab sence of sodium. Reduction of the second flavin species (presumably, covale ntly bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of similar to3.4 mM. This is v ery similar to the K-m for Na+ in the steady-state quinone reductase reacti on catalyzed by this enzyme. These data led us to conclude that the sodium- dependent step within the Na+-NQR is located between the noncovalently boun d FAD and the covalently bound FMN.