The aspartate-132 in subunit I (D(I-132)) of cytochrome c oxidase from Rhodobacter sphaeroides is located on the cytoplasmic surface of the protein at the entry point of a proton-transfer pathway used for both substrate and pumped protons (D-pathway). Replacement of D(I-132) by its nonprotonatable analogue asparagine (DN(I-132)) has been shown to result in a reduced overall activity of the enzyme and impaired proton pumping. The results from this study show that during oxidation of the fully reduced enzyme the reaction was inhibited after formation of the oxo-ferryl (F) intermediate (tau congruent with 120 microseconds). In contrast to the wild-type enzyme, in the mutant enzyme formation of this intermediate was not associated with proton uptake from solution, which is the reason the DN(I-132) enzyme does not pump protons. The proton needed to form F was presumably taken from a protonatable group in the D-pathway (e.g., E(I-286)), which indicates that in the wild-type enzyme the proton transfer during F formation takes place in two steps: proton transfer from the group in the pathway is followed by faster reprotonation from the bulk solution, through D(I-132). Unlike the wild-type enzyme, in which F formation is coupled to internal electron transfer from CuA to heme a, in the DN(I-132) enzyme this electron transfer was uncoupled from formation of the F intermediate, which presumably is due to the impaired charge-compensating proton uptake from solution. In the presence of arachidonic acid which has been shown to stimulate the turnover activity of the DN(I-132) enzyme (Fetter et al. (1996) FEBS Lett. 393, 155), proton uptake with a time constant of approximately 2 ms was observed. However, no proton uptake associated with formation of F (tau congruent with 120 micros) was observed, which indicates that arachidonic acid can replace the role of D(I-132), but it cannot transfer protons as fast as the Asp. The results from this study show that D(I-132) is crucial for efficient transfer of protons into the enzyme and that in the DN(I-132) mutant enzyme there is a "kinetic barrier" for proton transfer into the D-pathway.
Flash‐induced single‐electron reduction of cytochrome c oxidase. Compound F (oxoferryl state) by RuII(2,2'‐bipyridyl)2+ 3 [Nilsson (1992) Proc. Natl. Acad. Sci. USA 89, 6497‐6501] gives rise to three phases of membrane potential generation in proteoliposomes with τ values and contributions of ca. 45 μs (20%), 1 ms (20%) and 5 ms (60%). The rapid phase is not sensitive to the binuclear centre ligands, such as cyanide or peroxide, and is assigned to vectorial electron transfer from CuA to heme a. The two slow phases kinetically match reoxidation of heme a, require added H2O2 or methyl peroxide for full development, and are completely inhibited by cyanide; evidently, they are associated with the reduction of Compound F to the Ox state by heme a. The charge transfer steps associated with the F to Ox conversion are likely to comprise (i) electrogenic uptake of a ‘chemical’ proton from the N phase required for protonation of the reduced oxygen atom and (ii) electrogenic H+ pumping across the membrane linked to the F to Ox transition. Assuming heme a ‘electrical location’ in the middle of the dielectric barrier, the ratio of the rapid to slow electrogenic phase amplitudes indicates that the F to Ox transition is linked to transmembrane translocation of 1.5 charges (protons) in addition to an electrogenic uptake of one ‘chemical’ proton required to form Fe3+‐OH− from Fe4+ = O2−. The shortfall in the number of pumped protons and the biphasic kinetics of the millisecond part of the electric response matching biphasic reoxidation of heme a may indicate the presence of 2 forms of Compound F, reduction of only one of which being linked to full proton pumping.
SummaryActivation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apoforms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.
The ba 3 -type cytochrome c oxidase from T. thermophilus is phylogenetically very distant from the aa 3 -type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O 2 to water is catalysed at a haem a 3 -Cu B catalytic site. The three-dimensional structure of the ba 3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, R. sphaeroides and P. denitrificans aa 3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba 3 -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8*10 4 s −1 ) and formation of the peroxy (P R ) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7*10 4 s −1 , associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1100 s −1 , accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P R and F intermediate spectra are similar (Siletsky et al. (2007 and show that even though the architecture of the proton-conducting pathways is different in the ba 3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa 3 -type oxidases.
We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane.Even though CytcO forms a supercomplex with cytochrome bc 1 in the mitochondrial membrane, cyt. bc 1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory
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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