F 1-ATPase with cysteine instead of serine at residue 373 of the α subunit

Abstract

Escherichia coli strain AN718 contains the αS373F mutation in F 1F 0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F 1-ATPase activity. The revertant strain AN718SS2 containing the mutation αC373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F 1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in αS373F mutant F 1, were regained in αC373 F 1. N-Ethylmaleimide (NEM) inactivated αC373 f 1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of αC373 f 1 steady-state ATPase corresponded to incorporation of one NEM per f 1 (mol/mol), in just one of the three α subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue α373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F 1. The fact that NEM inhibits steady-state ATPase when only one α subunit of three is reacted suggests a cyclical catalytic mechanism.