Role of Subunit D in Ubiquinone-Binding Site of Vibrio cholerae NQR: Pocket Flexibility and Inhibitor Resistance

Abstract

The ion-pumping NADH: ubiquinone dehydrogenase (NQR) is a vital component of the respiratory chain of numerous species of marine and pathogenic bacteria, including Vibrio cholerae. This respiratory enzyme couples the transfer of electrons from NADH to ubiquinone (UQ) to the pumping of ions across the plasma membrane, producing a gradient that sustains multiple homeostatic processes. The binding site of UQ within the enzyme is an important functional and structural motif that could be used to design drugs against pathogenic bacteria. Our group recently located the UQ site in the interface between subunits B and D and identified the residues within subunit B that are important for UQ binding. In this study, we carried out alanine scanning mutagenesis of amino acid residues located in subunit D of V. cholerae NQR to understand their role in UQ binding and enzymatic catalysis. Moreover, molecular docking calculations were performed to characterize the structure of the site at the atomic level. The results show that mutations in these positions, in particular, in residues P185, L190, and F193, decrease the turnover rate and increase the Km for UQ. These mutants also showed an increase in the resistance against the inhibitor HQNO. The data indicate that residues in subunit D fulfill important structural roles, restricting and orienting UQ in a catalytically favorable position. In addition, mutations of these residues open the site and allow the simultaneous binding of substrate and inhibitors, producing partial inhibition, which appears to be a strategy used by Pseudomonas aeruginosa to avoid autopoisoning.

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function

“…It should be remembered that during catalytic turnover the subunits harboring the cofactors and the UQ-binding pocket need to undergo substantial conformational rearrangements that reduce the long distances for electron transfer (13); therefore the likelihood of the mutations having such indirect effects is high. More importantly, the UQ binding pocket proposed by Raba et al (42,43) is not only located between FMN NqrB (at the periplasmic surface) and riboflavin NqrB/E (at the cytoplasmic surface) but is also spatially closer to the former (~21 Å) than the latter (~36 Å) ( Figure S8). This positioning of the UQ head-ring seems difficult to reconcile with the current consensus on the electron transfer pathway in the enzyme (based on stopped-flow kinetics and cofactor-deletion mutants), in which electrons move from FMN NqrB to riboflavin NqrB/E , and from riboflavin NqrB/E to UQ (2,4,11,13).…”

“…We next discuss an alternative proposal about the catalytic binding position for the UQ head-ring in Na + -NQR. Based on alanine scanning mutagenesis of amino acid residues in membrane subunits NqrB and NqrD of V. cholerae Na + -NQR, Raba et al (42,43) proposed that the catalytic site for the UQ head-ring is located at the interface of transmembrane segments of NqrB and NqrD ( Figure S8), where the key residues forming the reaction pocket for the UQ head-ring and also for the quinolone ring of HQNO are Phe 185 and Phe 211 in TMHs 4 and 5 of NqrB, respectively, and Pro 185 , Leu 190 , and Phe 193 in TMH 6 of NqrD. In contrast, photoaffinity labeling studies using different photolabile UQs showed that the UQ head-ring binds to the NqrA subunit (7,14).…”

“…In contrast, photoaffinity labeling studies using different photolabile UQs showed that the UQ head-ring binds to the NqrA subunit (7,14). Since the studies by Raba et al (42,43) are largely based on Michaelis-Menten-type kinetic analyses of the activities of site-directed mutant enzymes, it is difficult to determine whether the proposed critical residues are directly involved in the interaction with the UQ ring or whether the mutations indirectly affect the UQ reaction by inducing longrange structural changes. It should be remembered that during catalytic turnover the subunits harboring the cofactors and the UQ-binding pocket need to undergo substantial conformational rearrangements that reduce the long distances for electron transfer (13); therefore the likelihood of the mutations having such indirect effects is high.…”

Electrostatics and water occlusion regulate covalently‐bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR

“…The electrons move downhill 31‐33 through a linear pathway (Figure 1C): two electrons from NADH are donated to FAD (located in subunit F); the electrons then move one by one to the 2Fe‐2S center in subunit F, to FMN C , to FMN B , and then are finally delivered to riboflavin, the final internal electron carrier. Riboflavin delivers the redox equivalents to ubiquinone, 28 which binds to subunit B 19,20,34‐36 …”

“…Riboflavin delivers the redox equivalents to ubiquinone, 28 which binds to subunit B. 19,20,[34][35][36] Strikingly, electron transfer during NQR catalysis is characterized by one-electron transitions. All physiological redox transitions for the two covalently-bond FMN and riboflavin molecules are one-electron transitions.…”