Glutamate 95 in NqrE Is an Essential Residue for the Translocation of Cations in Na-NQR.

The sodium-pumping NADH:quinone oxidoreductase (Na-NQR) is a bacterial enzyme that oxidizes NADH, reduces ubiquinone, and translocates Na across the membrane. We previously identified three acidic residues in the membrane-spanning helices, near the cytosol, NqrB-D397, NqrD-D133, and NqrE-E95, as candidates likely to be involved in Na uptake, and replacement of any one of them by a non-acidic residue affects the Na-dependent kinetics of the enzyme. Here, we have inquired further into the role of the NqrE-E95 residue by constructing a series of mutants in which this residue is replaced by amino acids with charges and/or sizes different from those of the glutamate of the wild-type enzyme.

Functional Studies on Membrane Proteins by Means of H/D Exchange in Infrared: Structural Changes in Na NQR from V. cholerae in the Presence of Lipids.

H/D exchange kinetics at the level of the amide proton in the mid infrared (1700-1500 cm) make it possible to study the conformational flexibility of membrane proteins, independent of size or the presence of detergent or lipids. Slow, medium, and fast exchanging domains are distinguished, which reveal a different accessibility to the solvent. Whereas amide hydrogens undergo rapid exchange with solvent in an open structure, hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent.

The conformational changes induced by ubiquinone binding in the Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit.

Na(+)-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin.

Infrared spectroscopic evidence of a redox-dependent conformational change involving ion binding residue NqrB-D397 in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a unique respiratory enzyme that conserves energy by translocating Na(+) through the plasma membrane. Found only in prokaryotes, the enzyme serves as the point of entry of electrons into the respiratory chain in many pathogens, including Vibrio cholerae and Yersinia pestis. In this study, a combined electrochemical and Fourier transform infrared (FTIR) spectroscopic approach revealed that Na(+)-NQR undergoes significant conformational changes upon oxidoreduction, depending on the monovalent cation present (Na(+), Li(+), K(+), or Rb(+)).

The role of glycine residues 140 and 141 of subunit B in the functional ubiquinone binding site of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone.

Thermodynamic contribution to the regulation of electron transfer in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is a fundamental enzyme of the oxidative phosphorylation metabolism and ionic homeostasis in several pathogenic and marine bacteria. To understand the mechanism that couples electron transfer with sodium translocation in Na(+)-NQR, the ion dependence of the redox potential of the individual cofactors was studied using a spectroelectrochemical approach. The redox potential of one of the FMN cofactors increased 90 mV in the presence of Na(+) or Li(+), compared to the redox potentials measured in the presence of other cations that are not transported by the enzyme, such as K(+), Rb(+), and NH(4)(+).

Electrochemical and infrared spectroscopic analysis of the interaction of the Cu(A) domain and cytochrome c(552) from Thermus thermophilus.

The hydrophobically guided complex formation between the Cu(A) fragment from Thermus thermophilus ba(3) terminal oxidase and its electron transfer substrate, cytochrome c(552), was investigated electrochemically. In the presence of the purified Cu(A) fragment, a clear downshift of the c(552) redox potential from 171 to 111mV±10mV vs SHE' was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions.