INVOLVEMENT OF HISTIDINE-91 OF THE BETA-SUBUNIT IN PROTON TRANSLOCATION BY THE PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA-COLI

The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out tran smembrane proton translocation coupled to transfer of a hydride equiva lent between NAD(+) and NADP(+). Mutations were made in histidine-91 o f the beta subunit of the pyridine nucleotide transhydrogenase of Esch erichia coli. This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is invo lved in proton translocation by the transhydrogenase. The mutant beta H91N retained 80% of the hydride transfer activity while proton transl ocation was reduced to 7%. This behavior is consistent with a role for beta His91 in the proton translocation pathway. Other mutations at th is residue affected the conformation of the enzyme. Thus, the enzyme i n mutants beta H91C, beta H91T, and beta H91S was unable to undergo th e conformational change that occurred on binding of the substrates NAD P(+) or NADPH. By contrast, the enzyme in the beta HB1K mutant was pre sent in the NADP(H)-induced conformation even in the absence of these substrates. Further evidence for the linkage between beta His91 and th e conformation of the beta subunit was obtained by labeling the transm embrane domain of the beta subunit with [C-14]N,N'-dicyclohexylcarbodi imide (DCCD). Labeling occurred most readily with the enzyme of beta H B1K. It is concluded that beta His91 is a component of the proton tran slocation pathway of the transhydrogenase and that its state of proton ation is probably linked to conformational changes induced by binding/ debinding of substrates during the catalytic cycle of the enzyme.