Mutation of Tyr(235) in the NAD(H)-binding subunit of the proton-translocating nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum affects the conformational dynamics of a mobile loop and lowers the catalytic activity of the enzyme

The Tyr residue in the mobile loop region of the soluble, domain I polypeptide (called Th-s) of the proton-translocating transhydrogenase from Rhodospirillum rubrum has been substituted by Asn and by Phe. The recombinant proteins were expressed at high levels in Escherichia coli and purified to homogeneity. The two well defined resonances at 6.82 and 7.12ppm, observed in the one-dimensional proton NMR spectrum of wild-type protein, and previously attributed to the Tyr residue, were absent in both mutants. In the Tyr(235) --> Phe mutant Th-s, they were replaced by two new resonances at 7.26 and 7.33 ppm, characteristic of a Phe residue. In both mutants, narrow resonances attributable to Met residues (and in the Tyr(235) --> Phe mutant, resonances attributable to Ala residues) were shifted relative to the wild type, but other features in the NMR spectra were unaffected. The conformational dynamics of the mobile loop closure in response to nucleotide binding by the protein were altered in the two mutants. The fluorescence emission from Trp(72) was unaffected by both Tyr substitutions, and the fluorescence was still quenched by NADH. The mutant Th-s proteins bound to chromatophore membranes depleted of their native Th-s with undiminished affinity. In these reconstituted systems, the K-m values for thio-NADP(+) and NADH, during light-driven transhydrogenation, were similar to those of wild-type, but the k(cat) values were decreased about 2-fold. In reverse transhydrogenation, the K-m values for NADPH were slightly decreased in the mutants relative to wild-type, but those for acetyl pyridine adenine dinucleotide were increased about 10- and 13-fold, respectively, and the k(cat) values were decreased about 2- and 5-fold, respectively, in the Tyr(235) --> Phe and Tyr(235) --> Asn mutants. It is concluded that Tyr(235) may contribute to the process of nucleotide binding and that substitution of this residue prevents proper functioning of the mobile loop in catalysis.