Inhibition by nitroso-chloramphenicol of the proton translocation in mitochondria

Abstract

We have found recently that, unlike chloramphenicol (CAP), its nitroreduction derivative nitroso-chloramphenicol (NO-CAP) behaved as a potent inhibitor of the energy-conserving mechanism in mitochondria [Abou-Khalil et al. Biochem. Pharmac. 29, 2605 (1980)]. Concentrations of 75 and 250 μM NO-CAP were required to inhibit ATP formation with glutamate and succinate, respectively, whereas similar CAP concentrations were without effect. Testing several key reactions associated with the biosynthesis of ATP, inhibitory concentrations of NO-CAP were found to interfere as follows: (a) the transport of an NAD-linked substrate (e.g. glutamate) into mitochondria was only partially inhibited, whereas that of an FAD-linked substrate (e.g. succinate) was not inhibited but was rather slightly activated; (b) the transport of P i was only inhibited at about 50%; (c) mitochondrial ADP transport was not affected at all; (d) the ATPase activity, measured either by Pi release in the presence of an uncoupler or by H + ejection, was only slightly affected; and (e) under either phosphorylation or no phosphorylation conditions and in the absence of P i, NO-CAP was found to completely block mitochondrial H + extrusion resulting from the oxidation of either succinate or glutamate; however, under similar conditions the oxidation of the two substrates was not totally inhibited. The possibility of interference by NO-CAP with reactive mitochondrial thiols groups is discussed in the light of previous data and current experiments showing protection by P i against NO-CAP effects on respiration. Moreover, NO-CAP as compared to conventional inhibitors of oxidative phosphorylation (e.g. rotenone, antimycin A, oligomycin, mersalyl and others) appeared to have a distinct mode of action on that process. The results demonstrate that the inhibitory effect of NO-CAP is primarily located at the respiratory chain level where the proton translocation activity is fully blocked.