Effect of Thiol Reagents on the Proton Conductivity of the H+-ATPase of Mitochondria

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Abstract
The role of thiol groups in the proton conduction by the H+‐ATPase of mitochondria is examined. A detailed kinetic analysis of the effect of arsenite and N‐ethylmaleimide on the anaerobic relaxation of the proton gradient set up by respiration in ‘inside‐out’ submitochondrial particles from beef‐heart has been carried out. Arsenite, which reacts with vicinal dithiols, is shown to enhance the proton conductivity of the H+‐ATPase. This effect is exerted on the F0 moiety of the complex and apparently mimics and is, in fact, favoured by a state of high proton conductivity induced in the complex by the respiratory Δμ+. N‐Ethylmaleimide (MalNEt), which is a permeant monothiol blocking reagent, appears to attack critical ‐SH groups in a reaction leading to inhibition of the proton conductivity of the H+‐ATPase. Also the inhibitory action of MalNEt on proton conduction is exerted on the F0 moiety of the H+‐ATPase. Whilst the stimulatory effect of arsenite develops rapidly, the inhibitory action of MalNEt is sluggish and takes more than 10 min to fully develop. This and other kinetic characteristics, as well a partial additivity of the inhibition by MalNEt with that by oligomycin, indicate that the inhibitory action of MalNEt is associated to a substantial conformational transition in F0. Differences in the mechanism of inhibition of proton conduction by MalNEt and triphenyltin are also presented.