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.     

Silver dichloroacetate: A compound with weak Ag-Cl bonding interactions and an extraordinary range of Cl NQR frequencies

Silver complexes of halocarbons and silver salts of halogenated organic acids (for example, silver chloroacetate) often show secondary Ag?X bonding interactions and unusually low ³⁵Cl NQR frequencies, due to secondary bonding of chlorines to silver atoms. The crystal structure of silver dichloroacetate has been determined at 100 K and shows six crystallographically-inequivalent chlorines. The structure is built from Ag₂(OOCCHCl₂)₂ dimers, similar to those found in silver chloroacetate; in both compounds the dimers are linked by additional Ag-O and Ag-Cl bonds. In the structure of silver dichloroacetate, two distinct conformations of the dichloromethyl groups are present. Two chlorines have no silver neighbors closer than 3.50 Å; two bridge to one Ag atom each, at Ag?Cl secondary bond distances of 2.8203(4) and 3.0196(4) Å, and two are apical, coordinating to at least two Ag neighbors each, at longer bond distances of 3.1401 (3)-3.3704(4) Å. Such very long distances are nevertheless shorter than the sum of the van der Waals radii of silver and chlorine, ca. 3.45 Å.The ³⁵Cl NQR spectrum of silver dichloroacetate at 77 K shows six signals scattered over the broad range from 35.600 to 38.498 MHz. Their EFG asymmetry parameters η were measured by the Fourier analysis of the slow beats in the spin echo envelope of the NQR signal of polycrystalline samples. The two highest-frequency chlorines have relatively low η values, 0.075 and 0.106, as befits Cl atoms not coordinated to Ag, and are placed by their conformations far from the carboxylate plane. The two middle-frequency chlorines have higher η values, 0.167 and 0.168, as expected for bridging Cl atoms. The two low-frequency chlorines have lower η values of 0.114 and 0.129, as expected for apical Cl atoms. For purposes of comparison, η values for Ag₂(OOCCH₂Cl)₂, Na(OOCCH₂Cl), and Ca(OOCCH₂Cl)₂ · H₂O were also recorded. So far, we have not observed any significant effect on the ³⁵Cl NQR parameters of halogenated organic anions coordinated to hard-acid metal ions (K⁺, Rb⁺, Ca²⁺). The effects of the different conformations of the Cl₂CH groups on the broad NQR frequency range are also discussed.     

NQR1 controls lifespan by regulating the promotion of respiratory metabolism in yeast

The activity and expression of plasma membrane NADH coenzyme Q reductase is increased by calorie restriction (CR) in rodents. Although this effect is well-established and is necessary for CR's ability to delay aging, the mechanism is unknown. Here we show that the Saccharomyces cerevisiae homolog, NADH-Coenzyme Q reductase 1 (NQR1), resides at the plasma membrane and when overexpressed extends both replicative and chronological lifespan. We show that NQR1 extends replicative lifespan in a SIR2-dependent manner by shifting cells towards respiratory metabolism. Chronological lifespan extension, in contrast, occurs via an SIR2-independent decrease in ethanol production. We conclude that NQR1 is a key mediator of lifespan extension by CR through its effects on yeast metabolism and discuss how these findings could suggest a function for this protein in lifespan extension in mammals.     

Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes

The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.