Abstract
The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the only respiratory enzyme that operates as a Na(+) pump. This redox-driven Na(+) pump is amenable to experimental approaches not available for H(+) pumps, providing an excellent system for mechanistic studies of ion translocation. An understanding of the internal electron transfer steps and their Na(+) dependence is an essential prerequisite for such studies. To this end, we analyzed the reduction kinetics of the wild type Na(+)-NQR, as well as site-directed mutants of the enzyme, which lack specific cofactors. NADH and ubiquinol were used as reductants in separate experiments, and a full spectrum UV-visible stopped flow kinetic method was employed. The results make it possible to define the complete sequence of redox carriers in the electrons transfer pathway through the enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMN(C) to its anionic flavosemiquinone state is the first Na(+)-dependent process, suggesting that reduction of this site is linked to Na(+) uptake. During the reduction reaction, two FMNs are transformed to their anionic flavosemiquinone in a single kinetic step. Subsequently, FMN(C) is converted to the flavohydroquinone, accounting for the single anionic flavosemiquinone radical in the fully reduced enzyme. A model of the electron transfer steps in the catalytic cycle of Na(+)-NQR is presented to account for the kinetic and spectroscopic data.
Highlights
Tion as the Hϩ-pumping NADH:quinone oxidoreductase, but there is nothing to indicate a common mechanism
In Naϩ-NQR electrons are carried through the enzyme by means of at least five redox cofactors: a noncovalently bound FAD and a 2Fe-2S center, in the NqrF subunit, where the binding site for NADH is located, two covalently attached FMNs in subunits B and C (FMNB and FMNC, respectively), and a noncovalently bound riboflavin that has not been definitely localized but that is believed to reside in the B or C subunit (7–12)
It is important to know the sequence of redox carriers along the path taken by electrons as they flow through the enzyme, as well as the kinetics and energetics of each step and how they are influenced by Naϩ and the electrical potential
Summary
Bacterial Strains and Growth Conditions—V. cholerae expressing the recombinant wild type Naϩ-NQR and mutants were grown as reported previously (1, 8, 14, 15). The flavin/protein molar ratios for wild type Naϩ-NQR, NqrB-T236Y, NqrC-T225Y, NqrB-T236Y/NqrC-T225Y, and NqrF-C76A were 3.6 Ϯ 0.2, 2.5 Ϯ 0.3, 2.8 Ϯ 0.4, 1.7 Ϯ 0.3, and 3.7 Ϯ 0.4 (7, 8). The six subunits (NqrA-F) are depicted together with the redox cofactors and the proposed pathway of electron flow through the enzyme (see “Discussion”). The scheme depicts the redox states of the cofactors after each kinetic phase in the reaction with NADH (without sodium), in the wild type enzyme, and in the cofactor mutants. At the top of the scheme, the redox cofactors, FAD, 2Fe-2S center, FMNC, FMNB, and riboflavin (RIB) are shown in the order of electron flow through the enzyme. The first component spectrum has minima at 460 and 400 nm This was previously assigned as two-electron reduction of a flavin molecule The second component spectrum has a minimum at 525 nm with shoulders 570 and 625 nm and a maximum at around 430
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