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 pol ypeptide (called Th-s) of the proton-translocating transhydrogenase fr om Rhodospirillum rubrum has been substituted by Asn and by Phe. The r ecombinant proteins were expressed at high levels in Escherichia coli and purified to homogeneity. The two well defined resonances at 6.82 a nd 7.12ppm, observed in the one-dimensional proton NMR spectrum of wil d-type protein, and previously attributed to the Tyr residue, were abs ent in both mutants. In the Tyr(235) --> Phe mutant Th-s, they were re placed by two new resonances at 7.26 and 7.33 ppm, characteristic of a Phe residue. In both mutants, narrow resonances attributable to Met r esidues (and in the Tyr(235) --> Phe mutant, resonances attributable t o Ala residues) were shifted relative to the wild type, but other feat ures in the NMR spectra were unaffected. The conformational dynamics o f the mobile loop closure in response to nucleotide binding by the pro tein were altered in the two mutants. The fluorescence emission from T rp(72) was unaffected by both Tyr substitutions, and the fluorescence was still quenched by NADH. The mutant Th-s proteins bound to chromato phore membranes depleted of their native Th-s with undiminished affini ty. In these reconstituted systems, the K-m values for thio-NADP(+) an d NADH, during light-driven transhydrogenation, were similar to those of wild-type, but the k(cat) values were decreased about 2-fold. In re verse transhydrogenation, the K-m values for NADPH were slightly decre ased in the mutants relative to wild-type, but those for acetyl pyridi ne adenine dinucleotide were increased about 10- and 13-fold, respecti vely, and the k(cat) values were decreased about 2- and 5-fold, respec tively, in the Tyr(235) --> Phe and Tyr(235) --> Asn mutants. It is co ncluded that Tyr(235) may contribute to the process of nucleotide bind ing and that substitution of this residue prevents proper functioning of the mobile loop in catalysis.