Interaction of Dicyclohexylcarbodiimide with the Proton‐Conducting Pathway of Mitochondrial H+‐ATPase

Abstract

In this paper a study is presented of the interaction of N,N′‐dicyclohexylcarbodiimide (DCCD) with the H+‐ATPase of mitochondria. Inhibition of proton conduction by the membrane sector (Fo) of the H+‐ATPase has been characterized by kinetics analysis of the effect of DCCD on the anaerobic decay of ΔμH+ set up by respiration in submitochondrial particles containing (ESMP) or deprived (USMP) of the catalytic sector (F1).The inhibitory effect of DCCD was directly related to the amount of DCCD bound independently of the time of incubation and of the concentration applied. Both in ESMP or USMP 2—3 nmol DCCD/mol Fo were required for the maximal inhibition of proton conduction.In ESMP the anaerobic relaxation of ΔμH+ is biphasic. Both phases were inhibited by DCCD and the titration curve indicated that the slow phase is directly related to the hydrolytic activity of H+‐ATPase. In USMP the anaerobic relaxation of ΔμH+ is monophasic. DCCD, at low concentrations, restored the biphasic pattern with the appearance of a transient fast phase having a kinetic constant higher than that of the control. Whilst the slow phase was immediately inhibited by DCCD the fast phase started to be inhibited only at concentrations higher than 1 nmol DCCD bound/mg protein.The data reported support the view that the fast phase of proton conduction reflects a conformational state of Fo, with higher proton conductivity, which is induced by high aerobic ΔμH+.Electrophoretic analysis of the labeling by [14C]DCCD of membrane polypeptides indicated that the inhibition of the state of low proton conductivity of Fo can be correlated with binding to the 8000‐Mr form of DCCD‐binding proteolipid, whilst inhibition of the state of high proton conductivity of Fo can be correlated with binding to the 16000‐Mr polypeptide.