Glutamine 132 in the NAD(H)-Binding Component of Proton-Translocating Transhydrogenase Tethers the Nucleotides before Hydride Transfer

• Published in: Biochemistry (Easton) 2003-02, Vol.42 (5), p.1217-1226
• Main Authors: van Boxel, Gijs I, Quirk, Philip G, Cotton, Nick P. J, White, Scott A, Jackson, J. Baz
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Asparagine - genetics; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Binding Sites - genetics; Crystallization; Crystallography, X-Ray; Electron Transport - genetics; Glutamine - chemistry; Glutamine - genetics; Kinetics; Mutagenesis, Site-Directed; NAD - chemistry; NAD - genetics; NADP - chemistry; NADP Transhydrogenases - antagonists & inhibitors; NADP Transhydrogenases - chemistry; NADP Transhydrogenases - genetics; Protons; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Rhodospirillum rubrum - enzymology; Rhodospirillum rubrum - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi027032e

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)2dIII1. The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)2dIII1 was