PREDICTION AND SITE-SPECIFIC MUTAGENESIS OF RESIDUES IN TRANSMEMBRANE ALPHA-HELICES OF PROTON-PUMPING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASES FROM ESCHERICHIA-COLI AND BOVINE HEART-MITOCHONDRIA

Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD. The complete gene for this transhydrogenase was constructed, and the protein primary structure was determined from the cDNA. As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala591 (previously Phe), Val777 (previously Glu), and Ala782 (previously Arg). The Escherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD. Alignment of the protein primary structure of the bovine trashydrogenase with that of the transhydrogenase from E. coli showed an identity of 52%, indicating similarly folded structures. Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of the revised bovine heart and E. coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases. In E. coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta subunit. Various conserved amino acid residues of the E. coli transhydrogenase located in or close to predicted transmembrane alpha-helixes were replaced by site-specific mutagenesis. Conserved negatively charged residues in predicted transmembrane alpha-helices possibly participating in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) located close to the predicted alpha-helices 7 and 9 of the beta subunit. beta Glu82 was replaced by Lys or Gin and beta Asp213 by Asn or His. However, the catalytic as well as the proton pumping activity was retained. In constrast, mutagenesis of the conserved beta His91 residue (His664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially inactive enzyme. Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abolishing proton pumping. Since no other conserved acidic or basic residues were predicted in transmembrane alpha-helices regardless of the prediction algorithm used, proton translocation by transhydrogenase was concluded to involve a basic rather than an acidic residue. The only conserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), located in the predicted a-helix 10 of the E. coli transhydrogenase, previously suggested to function as a redox-active dithiol, proved not to be essential, suggesting that redox-active dithiols do not play a role in the mechanism of transhydrogenase.