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 signals 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 was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.
Bogachev
A V
AV
Department of Bioenergetics, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
Bertsova
Y V
YV
Barquera
B
B
Verkhovsky
M I
MI
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D017494:Proton Pumps / Q000378:metabolism; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D013053:Spectrophotometry; D014733:Vibrio / Q000201:enzymology*
D007506:Iron-Sulfur Proteins; D017494:Proton Pumps; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D012964:Sodium; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases