PROPERTIES OF THE SOLUBLE POLYPEPTIDE OF THE PROTON-TRANSLOCATING TRANSHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM OBTAINED BY EXPRESSION IN ESCHERICHIA-COLI

Transhydrogenase, which catalyses the reduction of NADP(+) by NADH cou pled to proton translocation across a membrane, may be unique in the p hotosynthetic bacterium Rhodospirillum rubrum. Unlike the homologous e nzyme from animal mitochondria and other bacterial sources, it has a w ater-soluble polypeptide, which exists as a dimer (Th-s), that can be reversibly dissociated from the membrane component [Williams, R., Cott on, N. P. J., Thomas, C. M. and Jackson, J. B. (1994) Microbiology, 14 0, 1595-1604]. We have expressed the gene for Th-s in cells of Escheri chia coli under control of the tac promoter and a strong ribosome bind ing site. The protein, purified by column chromatography, fully recons tituted transhydrogenation activity to everted membrane vesicles of Rh s. rubrum that had been washed to remove Th-s. The purifed expressed p rotein was prepared in quantities over 100-fold greater than were obta ined from wild-type Rhs. rubrum. The fluorescence spectrum of purified expressed Th-s had an intense and unusually short wavelength emission maximum at 310 nm with shoulders at 298 and 322 nm. Time-resolved mea surements indicated that the fluorescence decay was almost monoexponen tial with a lifetime of 5.2 ns. On denaturation with 4 M guanidine hyd rochloride, the emission band shifted to 352 nm and decreased in inten sity. In the native protein, the fluorophore was relatively inaccessib le to quenching solutes, such as iodide ions and acrylamide. It is con cluded that the fluorescence emission arises mainly from the single tr yptophan residue of Th-s (Trp72), which is locked into a rigid conform ation and is located in a highly non-polar environment. The 310-nm flu orescence of Th-s was quenched by NADH, maximally to 46 %, The apparen t binding constant was 18 mu M. The fluorescence of Th-s-bound NADH wa s enhanced relative to the nucleotide in free solution and its emissio n maximum was shifted to a shorter wavelength (440 nm). These data sup port previous indications that the NADH binding site is located in dom ain I of proton-translocating transhydrogenase. Excitation of Th-s at 280 nm did not lead to sensitized emission at 440 nm from bound NADH. This indicates that the quenching of fluorescence of Th-s by NADH does not result from resonance energy transfer from Trp72 to the bound nuc leotide. NAD(+), NADP(+) and NADPH had little effect on the protein fl uorescence. The kinetics of quenching of Th, fluorescence by NADH were examined after mixing in a stopped-flow device. The 'on' rate constan t for nucleotide binding was approximately 8 X 10(6) M(-1) s(-1) and t he 'off' constant approximately 150 s(-1).