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. All of the mutants showed altered steady-state kinetics with the acceleration of turnover by Na+ greatly diminished. Selected mutants were studied by other physical methods. Membrane potential measurements showed that NqrE-E95D and A are significantly less efficient in ion transport. NqrE-E95A, Q, and D were studied by transient kinetic measurements of the reduction of the enzyme by NADH. In all three cases, the results indicated inhibition of the electron-transfer step in which the FMNC becomes reduced. This is the first Na+-dependent step and is associated with Na+ uptake by the enzyme. Electrochemical measurements on NqrE-E95Q showed that the Na+ dependence of the redox potential of the FMN cofactors has been lost. The fact that the mutations at the NqrE-E95 site have specific effects related to translocation of Na+ and Li+ strongly indicates a definite role for NqrE-E95 in the cation transport process of Na+-NQR.

The obligate respiratory supercomplex from Actinobacteria.

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.

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. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na(+)-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na(+)-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.

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(+)). In the presence of the inhibitor Rb(+), additional conformational changes are evident, indicating a changed accessibility of the sodium binding sites. In electrochemically induced FTIR difference spectra, the involvement of deprotonated acid residues in the binding of cations, together with the spectral features, that point toward a monodentate binding mode for these acid residues in the oxidized form of the enzyme and bidentate binding in the reduced form could be identified. The measurements confirmed that NqrB-D397 is one of the acid residues involved in Na(+) and Li(+) binding. In the NqrB-D397E mutant, the spectral features characteristic of COO(-) groups are shifted, and a weakening of the hydrogen binding of the ion binding cluster is revealed. Finally, H-D exchange kinetics of amide protons confirmed that Na(+)-NQR adopts different conformations, with different accessibilities to the aqueous environment, depending on the cation present, which contributes to the selectivity mechanism of ion translocation.

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. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na(+)-NQR with its electron acceptor, ubiquinone.

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. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

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)(+). This shift in redox potential of one FMN confirms the crucial role of the FMN anionic radicals in the Na(+) pumping mechanism and demonstrates that the control of the electron transfer rate has both kinetic (via conformational changes) and thermodynamic components.

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-1) 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. Here, we describe the protocol that was used to study the effect of phospholipids on the overall structure of the Na+ NQR from V. cholerae, a sodium pumping membrane protein.