Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase

Abstract

Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues. This enzyme contains seven cysteines, of which five are located in the α subunit and two are in the β subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (αC292T, αC339T, αC395S, αC397T, αC435S, βC147S, βC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the β subunit to a trypsin-sensitive state. Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities. Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities. The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes. No bound NADP(H) was detected in the enzymes. Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this process. Replacement of cysteine residues in the α subunit resulted in no or little change in activity, suggesting that the basis for the decreased activity was probably the modification of the conserved β-subunit residue Cys-260 or (less likely) the non-conserved β-subunit residue Cys-147. It is concluded that the cysteine-free transhydrogenase is structurally and mechanistically very similar to the wild-type enzyme, with minor modifications of the properties of the NADP(H) site, possibly mediated by the βC260S mutation. The cysteine-free construct will be a valuable tool for studying structure–function relationships of transhydrogenases.