Is cytochrome b glutamic acid 272 a quinol binding residue in the bc1 complex of Saccharomyces cerevisiae?

The mitochondrial bc1 complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c coupled to a vectorial translocation of protons across the membrane. On the basis of the three-dimensional structures of the bc1 complex in the presence of the inhibitor stigmatellin, it was assumed that the substrate quinol binding involves the cyt b glutamate residue E272 and the histidine 181 on the Rieske protein. Although extensive mutagenesis of glutamate E272 has been carried out, different experimental results were recently obtained, and different conclusions were drawn to explain its role in the bifurcated electron/proton transfer at the QO site. This residue is not totally conserved during evolution. We show in this study that replacement of E272 with apolar residues proline and valine naturally present in some organisms did not abolish the bc1 activity, although it slowed down the kinetics of electron transfer. The Km value for the binding of the substrate quinol was not modified, and the EPR data showed that the quinone/quinol binding still occurred in the mutants. Binding of stigmatellin was retained; however, mutations E272P,V induced resistance toward the QO site inhibitor myxothiazol. The pH dependence of the bc1 activity was not modified in the absence of the glutamate E272. Our results suggest that this residue may not be involved in direct substrate binding or in its direct deprotonation. Revertants were selected from the respiratory deficient mutant E272P. The observed suppressor mutations introduced polar residues serine and threonine at position 272. The data lead us to suggest that E272 may be involved in a later step on the proton exit pathway via the interaction with a water molecule.

Ascorbate stimulates monooxygenase-dependent steroidogenesis in adrenal zona glomerulosa.

It is well known that ascorbic acid (Asc) is highly concentrated in the adrenal gland, but its function in the gland is not thoroughly elucidated. We therefore examined the possibility that Asc participates in steroidogenic monooxygenase systems of the adrenal cortex with the aid of the regenerating system including outer mitochondrial membrane cytochrome b (OMb). When Asc availability was limited in rat mutants unable to synthesize Asc, the increase in plasma aldosterone concentration under Na-deficiency was suppressed without effect on plasma corticosterone concentration. Aldosterone formation in the isolated mitochondrial fraction of the zona glomerulosa (zG) of the adrenal cortex was stimulated by the addition of Asc and NADH, while corticosterone formation was not. Consistently zG showed a high level of Asc regeneration activity and was rich in OMb among adrenocortical zones. Taken together, the enhanced aldosterone formation that is catalyzed by one of the steroidogenic monooxygenases, P450aldo, may be supported by Asc with its regenerating system.

Membrane-associated maturation of the heterotetrameric nitrate reductase of Thermus thermophilus.

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme.

Screening for mutations related to atovaquone/proguanil resistance in treatment failures and other imported isolates of Plasmodium falciparum in Europe.

BACKGROUND: Two single-point mutations of the Plasmodium falciparum cytochrome b gene (Tyr268Asn and Tyr268Ser) were recently reported in cases of atovaquone/proguanil (Malarone) treatment failure. However, little is known about the prevalence of codon-268 mutations and their quantitative association with treatment failure. METHODS: We set out to assess the prevalence of codon-268 mutations in P. falciparum isolates imported into Europe and to quantify their association with atovaquone/proguanil treatment failure. Isolates of P. falciparum collected by the European Network on Imported Infectious Disease Surveillance between April 2000 and August 2003 were analyzed for codon-268 mutations, by use of polymerase chain reaction-restriction fragment-length polymorphism. RESULTS: We successfully screened 504 samples for the presence of either Tyr268Ser or Tyr268Asn. One case of Ser268 and no cases of Asn268 were detected. Therefore, we can be 95% confident that the prevalence of Ser268 in the European patient pool does not exceed 0.96% and that Asn268 is less frequent than 0.77%. In 58 patients treated with atovaquone/proguanil, Tyr268Ser was present in 1 of 5 patients with treatment failure but in 0 of 53 successfully treated patients. CONCLUSIONS: Tyr268Ser seems to be a sufficient, but not a necessary, cause for atovaquone/proguanil treatment failure. The prevalence of both codon-268 mutations is currently unlikely to be >1% in the European patient pool.

Decreased neointimal formation in Nox2-deficient mice reveals a direct role for NADPH oxidase in the response to arterial injury.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced, in part, from NADPH oxidase in response to host invasion and tissue injury. Defects in NADPH oxidase impair host defense; however, the role of ROS and RNS in the response to tissue injury is not known. We addressed this issue by subjecting leukocyte oxidase (Nox2)-deficient (Nox2-/-) mice to arterial injury. Femoral artery injury was associated with increased Nox2 expression, ROS/RNS production, and oxidative protein and lipid modification in wild-type mice. In Nox2-/- mice, RNS-mediated protein oxidation, as monitored by protein nitrotyrosine content, was significantly diminished. This was accompanied by reduced neointimal proliferation, as monitored by intimal thickness and intimal/medial ratio, in Nox2-/- compared to wild-type mice. In addition, Nox2 deficiency led to reduced cellular proliferation and leukocyte accumulation. These data indicate that Nox2-mediated oxidant production has a requisite role in the response to tissue injury.

gp91phox-containing NADPH oxidase mediates endothelial dysfunction in renovascular hypertension.

BACKGROUND: Isoforms of the NADPH oxidase contribute to vascular superoxide anion (*O2-) formation and limit NO bioavailability. We hypothesized that the endothelial gp91phox-containing NADPH oxidase is predominant in generating the O2- to scavenge endothelial NO and thus is responsible for the development of endothelial dysfunction. METHODS AND RESULTS: Endothelial dysfunction was studied in aortic rings from wild-type (WT) and gp91phox-knockout (gp91phox-/-) mice with and without renovascular hypertension induced by renal artery clipping (2K1C). Hypertension induced by 2K1C was more severe in WT than in gp91phox-/- mice (158+/-2 versus 149+/-2 mm Hg; P<0.05). Endothelium-dependent relaxation to acetylcholine (ACh) was attenuated in rings from clipped WT but not from clipped gp91phox-/- mice. The reactive oxygen species (ROS) scavenger Tiron, PEG-superoxide dismutase, and the NADPH oxidase inhibitory peptide gp91ds-tat enhanced ACh-induced relaxation in aortae of clipped WT mice. Inhibition of protein kinase C, Rac, and the epidermal growth factor receptor kinase, elements involved in the activation of the NADPH oxidase, restored normal endothelium-dependent relaxation in vessels from clipped WT mice but had no effect on relaxations in those from gp91phox-/- mice. Relaxations to exogenous NO were attenuated in vessels from clipped WT but not clipped gp91phox-/- mice. After removal of the endothelium or treatment with PEG-superoxide dismutase, NO-induced relaxations were identical in vessels from clipped and sham-operated WT and gp91phox mice. CONCLUSIONS: These data indicate that the formation of O2- by the endothelial gp91phox-containing NADPH oxidase accounts for the reduced NO bioavailability in the 2K1C model and contributes to the development of renovascular hypertension and endothelial dysfunction.

Functional respiratory chain analyses in murid xenomitochondrial cybrids expose coevolutionary constraints of cytochrome b and nuclear subunits of complex III.

The large number of extant Muridae species provides the opportunity of investigating functional limits of nuclear/mitochondrial respiratory chain (RC) subunit interactions by introducing mitochondrial genomes from progressively more divergent species into Mus musculus domesticus mtDNA-less (rho0) cells. We created a panel of such xenomitochondrial cybrids, using as mitochondrial donors cells from six murid species with divergence from M. m. domesticus estimated at 2 to 12 Myr before present. Species used were Mus spretus, Mus caroli, Mus dunni, Mus pahari, Otomys irroratus, and Rattus norvegicus. Parsimony analysis of partial mtDNA sequences showed agreement with previous molecular phylogenies, with the exception that Otomys did not nest within the murinae as suggested by some recent nuclear gene analyses. Cellular production of lactate, a sensitive indicator of decreased respiratory chain ATP production, correlated with divergence. Functional characterization of the chimeric RC complexes in isolated mitochondria using enzymological analyses demonstrated varying decreases in activities of complexes I, III, and IV, which have subunits encoded in both mitochondrial and nuclear genomes. Complex III showed a striking decline in electron transfer function in the most divergent xenocybrids, being greatly reduced in the Rattus xenocybrid and virtually absent in the Otomys xenocybrid. This suggests that nuclear subunits interacting with cytochrome b face the greatest constraints in the coevolution of murid RC subunits. We sequenced the cytochrome b gene from the species used to identify potential amino acid substitutions involved in such interactions. The greater sensitivity of complex III to xenocybrid dysfunction may result from the encoding of redox center apoproteins in both nuclear and mitochondrial genomes, a unique feature of this RC complex.

Effect of the systemic fungicide carboxin on electron transport function in membranes of Micrococcus denitrificans.

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) inhibited oxidation of succinate by membranes prepared from Micrococcus denitrificans, the K(i) being 16 muM. Oxycarboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4,4-dioxide), F831 (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4-oxide), and another succinate oxidase inhibitor, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTB) were less effective inhibitors of succinate oxidation by membranes of M. denitrificans. Oxidation of other substrates (nicotinamide adenine dinucleotide, reduced form, d-lactate, l-lactate, malate, and d,l-alpha-hydroxybutyrate) was inhibited to a lesser degree by carboxin, and formate oxidation was entirely resistant. With all substrates tested, oxycarboxin, the dioxide analogue of carboxin, was less effective than carboxin. Carboxin also inhibited dichlorophenol indophenol (DCIP) reductase activities by these membranes in a manner both qualitatively and quantitatively similar to the inhibition of oxidation of the various substrates. The inhibition of DCIP reductase activities by TTB was qualitatively similar to carboxin, but TTB was a less effective inhibitor with all substrates tested. The inhibition of DCIP reductase by carboxin could be relieved by phenazine methosulfate with all substrates except d-lactate. Only slight inhibition of d-lactate-stimulated uptake of [(14)C]glycine by these membrane vesicles was seen with carboxin. Uptake of [(14)C]glycine could be stimulated to varying degrees with the other substrates tested, but in no case did carboxin cause significant inhibition. Membranes isolated from M. denitrificans are a useful system for investigating the mechanism of inhibition of electron transport function by carboxin, and the use of this system for evaluations of carboxin and its metabolites is suggested.