The three-dimensional structure of cytochrome coxidase (COX) reveals two potential input proton channels connecting the redox core of the enzyme with the negatively charged (N-) aqueous phase. These are denoted as the K-channel (for the highly conserved lysine residue, K362 in Rhodobacter sphaeroides COX) and the D-channel (for the highly conserved aspartate gating the channel at the N-side, D132 in R. sphaeroides). In this paper, it is shown that the K362M mutant form of COX from R. sphaeroides, although unable to turnover with dioxygen as electron acceptor, can utilize hydrogen peroxide as an electron acceptor, with either cytochrome c or ferrocyanide as electron donors, with turnover that is close to that of the wild-type enzyme. The peroxidase activity is similar to that of the wild-type oxidase and is coupled to the generation of a membrane potential and to proton pumping. In contrast, no peroxidase activity is revealed in the D-channel mutants of COX, D132N, and E286Q. Reduction by dithionite of heme a3 in the fully oxidized oxidase is severely inhibited in the K362M mutant, but not in the D132N mutant. Apparently, mutations in the D-channel arrest COX turnover by inhibiting proton uptake associated with the proton-pumping peroxidase phase of the COX catalytic cycle. In contrast, the K-channel appears to be dispensable for the peroxidase phase of the catalytic cycle, but is required for the initial reduction of the heme-copper binuclear center in the first half of the catalytic cycle.

2-n-Heptyl 4-hydroxyquinoline-N-oxide (HOQNO) inhibits the succinate: quinone oxidoreductase activity of isolated and membrane-bound succinate:menaquinone oxidoreductase of B. subtilis. The inhibition pattern resembles closely that observed for a-thenoyltrifluoroacetone and carboxins in the mitochondrial succinate:ubiquinone oxidoreductase: ca. 90% of the activity is highly sensitive to HOQNO (K; ca. 0.2 pM for the isolated enzyme) whereas the rest 10% proves to be resistant to the inhibitor. HOQNO binding is shown to perturb the absorption spectrum of the ferrous di-heme cytochrome b of the B. subtilis succinate:quinone oxidoreductase both in the a and Sorer bands. In addition, the inhibitor is shown to bring about a negative shift of Em of the low-potential heine b. It is suggested that HOQNO interacts with a menasemiquinone binding site near the low-potential heine and suppresses the MQ'--to-MQH2 step of the quinone reductase reaction but allows partly for the MQto-MQ'-transition to occur; dismutation of MQ" formed in the latter reaction to MQ and MQH 2 may account for the 10% of the enzyme activity insensitive to HOQNO.Key words: 2-n-Heptyl 4-hydroxyquinoline-N-oxide; Succinate quinone reductase; Cytochrome b, Bacillus subtilis; Menasemiquinone; Complex II known to be blocked specifically by a c~-thenoyltrifluoroacetone (TTFA) and carboxins [1,5,6]. These inhibitors are believed to act at the ubiquinone (UQ) reduction site, interrupting electron transfer between the high-potential iron-sulfur centre S-3 and UQ [1,7] and destabilizing the tightly bound ubisemiquinone radical [8][9][10][11]. B. subtilis SQR is not sensitive to these compounds but is inhibited by n-heptyl 4-hydroxyquinoline-Noxide (HOQNO) [4,12]. HOQNO is a potent UQ and menaquinone (MQ) antagonist acting on many respiratory cytochrome b containing quinone-reactive redox enzymes in various organisms. In particular, HOQNO is known as a classical inhibitor of the mitochondrial cytochrome bct complex [13], binding at the quinone reductase site of this enzyme (so-called, centre i) and bringing about a spectral perturbation of the high-potential heme b [14] and a positive shift of its Em [15].In this work we have studied effects of HOQNO on the purified and membrane-bound B. subtilis SQR. The HOQNO inhibition pattern is rather similar to the effect of TTFA and carboxins on the mitochondrial SQR. In addition HOQNO perturbs the optical absorption spectrum of cytochrome b in SQR and brings about a negative shift of the low-potential heme (heme bL) of the cytochrome. Implications of these findings for the mechanism of HOQNO inhibitory action are discussed.

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At a pH of <7, respiration of Bacillus subtilis cells on endogenous substrates shut down almost completely upon addition of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone [CCCP]) and a K ؉ -ionophore (valinomycin). The same effect was observed with cell spheroplasts lacking the cell wall. The concentration of CCCP required for 50% inhibition of the endogenous respiration in the presence of K ؉ -valinomycin was below 100 nM. Either CCCP or valinomycin alone was much less efficient than the combination of the two. The inhibitory effect was easily reversible and depended specifically on the H ؉ and K ؉ concentrations in the medium. Similar inhibition was observed with respect to the reduction of the artificial electron acceptors 2,6-dichlorophenolindophenol (DCPIP) and N,N,N,N-tetramethyl-p-phenylenediamine cation (TMPD ؉ ), which intercept reducing equivalents at the level of menaquinol. Oxidation of the reduced DCPIP or TMPD in the bacterial cells was not sensitive to uncoupling. The same loss of the electron transfer activities as induced by the uncoupling was observed upon disruption of the cells during isolation of the membranes; the residual activities were not further inhibited by the uncoupler and ionophores. We conclude that the menaquinonedependent electron transfer in the B. subtilis respiratory chain is facilitated, thermodynamically or kinetically, by membrane energization. A requirement for an energized state of the membrane is not a specific feature of succinate oxidation, as proposed in the literature, since it was also observed in a mutant of B. subtilis lacking succinate:quinone reductase as well as for substrates other than succinate. Possible mechanisms of the energy-dependent regulation of menaquinone-dependent respiration in B. subtilis are discussed.There is a classical phenomenon of a so-called respiratory control in mitochondrial oxidative phosphorylation (35; reviewed in reference 18). Electron flow in the mitochondrial respiratory chain becomes slow as the membrane is energized (the state of respiratory control) and can be stimulated under these conditions by addition of ADP, which turns on ATP synthesis, or by the uncouplers of oxidative phosporylation, which dissipate the transmembrane electrochemical proton potential difference (⌬H ϩ ). Although described initially for mitochondria, the effect of respiratory control is often assumed to apply to bacterial coupled respiration as well (see, e.g., reference 43).It is therefore interesting that respiration of the aerobic bacterium Bacillus subtilis is stimulated rather than suppressed by proton motive force. The fact that the respiration of B. subtilis cells can be inhibited by K ϩ /H ϩ and Na ϩ /H ϩ antiporters and protonophores was noticed about 10 years ago (3, 37, 46) and confirmed more recently by Shirawski and Unden (47). A potentially relevant observation is that the succinate oxidase activity of B. subtilis cells decreases drastically during isolation of the membranes (36, 47); this effect could also be a consequence of memb...