Redox Properties of the Prosthetic Groups of Na+-Translocating NADH:Quinone Oxidoreductase. 1. Electron Paramagnetic Resonance Study of the Enzyme

Redox properties of all EPR-detectable prosthetic groups of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E-m = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E-m(1) = -190 mV and E-m(2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC Subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E-m = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E-m = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB Subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na+-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 +/- 1.5 angstrom, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.