A characteristic feature of the vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) of plant cells is its high sensitivity to irreversible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl reagents. Previous investigations in this laboratory have demonstrated that the primary site for substrate-protectable covalent modification of the V-PPase by 14C-labeled NEM maps to a single M(r) 14,000 V8 protease fragment (V8(14)K) (Zhen, R.-G., Kim, E. J., and Rea, P. A. (1994) J. Biol. Chem. 269, 23342-23350). Here, we describe site-directed mutagenesis of the cDNA encoding the V-PPase from Arabidopsis thaliana, its heterologous expression in Saccharomyces cerevisiae and single substitution of all 9 conserved Cys residues to either Ser or Ala. In all cases, except one, Cys mutagenesis exerts little or no effect on either the catalytic activity or susceptibility of the enzyme to inhibition by NEM. By contrast, and in complete agreement with the results of peptide mapping experiments, substitution of Cys634, the sole conserved cysteine residue encompassed by V8(14)K, with Ser or Ala generates enzyme that is insensitive to NEM but active in both PPi hydrolysis and PPi-dependent H+ translocation. The specific requirement for Cys634 for inhibition by NEM and the dispensability of all of the conserved Cys residues, including Cys634, for V-PPase function indicate that the inhibitory action of maleimides reflects steric constraints imposed by the addition of a substituted alkyl group to the side chain of Cys634 rather than direct participation of this amino acid residue in catalysis.