Sequencing and Preliminary Characterization of the Na⁺-Translocating NADH:Ubiquinone Oxidoreductase from Vibrio harveyi

Abstract

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.

Riboflavin is a component of the Na + -pumping NADH–quinone oxidoreductase from Vibrio cholerae

“…A similar analysis showed that riboflavin is present also in Na ϩ -NQR purified from the bacterium Vibrio harveyi (ref. 9 and data not shown), which demonstrates that the riboflavin cofactor is not limited to the enzyme from V. cholerae.…”

“…Recombinant Na ϩ -NQR from V. cholerae was purified according to a protocol described before (10). The V. harveyi Na ϩ -NQR was prepared as described (9).…”

“…Na ϩ -NQR is the entry point for electrons into the respiratory chain of a number of marine and pathogenic bacteria (5-7). The enzyme has been isolated from several of these bacteria (8,9) including Vibrio cholerae (10). The nqr operon from V. cholerae has been cloned and expressed in the parent organism (10), and the purified recombinant enzyme has been used in the current studies.…”

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

“…A recent study of NosR from Paracoccus denitrificans shows agreement with the described topology (R. J. M. van Spanning, The flavin-binding domain of NosR is found in, among other proteins, the NqrC subunit of the Na ϩ -translocating NADH: quinone oxidoreductase of the genus Vibrio. NqrC was shown to carry FMN covalently bound as a phosphodiester to a threonine residue (17,27,52). Given the spectral evidence and the supporting flavin-binding consensus sequence, we suggest that NosR of P. stutzeri carries a flavin cofactor covalently bound to T163.…”

NADH Oxidation by the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

“…In addition, motifs for the binding of flavin and NADH were identified on NqrF (14). Subunits NqrB and NqrC each contain one FMN that is covalently linked to a threonine residue (15)(16)(17). Recently, riboflavin was identified as a component of the Na ϩ -NQR complex from V. cholerae (18).…”