Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.

The purpose of this study was to generate, by real-time PCR, a quantitative expression level profile of the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A and 2B, in 26 adult and 3 fetal tissues, for better understanding of their roles in xenobiotic and endobiotic metabolism. Adult liver contained the highest level of combined UGTs mRNA, and UGT2B4 was the most abundant isoform in this tissue (40% of total). Other well expressed hepatic UGTs, in decreasing order of mRNA level, were 1A9, 2B7, 1A4, 2B10, 1A1, 1A6, 2B11, 2B15, 1A3, 2A3, 2B17 and 2B28. UGT2B4 was by far the most abundant isoform in the fetal liver (90% of total). The combined UGT mRNA expression in both adult and fetal olfactory epithelium was high, about 20% the adult hepatic level. Interestingly, a large developmental change was found in this tissue from high UGT2A1 and UGT2A2 expression in the fetus to UGT1A6 in the adult. The most abundantly expressed UGTs in the small intestine were 2A3, 1A10, 1A1, 1A6 and 2B7, while 1A10 and 2A3 predominated in the colon. The results provide the most comprehensive data to date on the tissue distribution of the human UGTs.

J. Biol. Chem. 260, 2458 -2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c 552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.

Spheroplasts from aerobically grown wild‐type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc 1 complex by myxothiazol. Coupled to this activity, which involves only b‐type cytochromes, there is translocation of 1.5–1.9 H+/e− across the cytoplasmic membrane. Similar H+ translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa 3 to which it is structurally related.

ABSTRACT:The glucuronidation of 17␤-estradiol (␤-estradiol) and 17␣-estradiol (epiestradiol) was studied to elucidate how the orientation of the 17-OH group affects conjugation at the 3-OH or the 17-OH of either diastereomer. Recombinant human UDP-glucuronosyltransferases (UGTs) UGT1A1, UGT1A3, UGT1A7, UGT1A8, and UGT1A10 conjugated one or both diastereomers, mainly at the 3-OH. The activity of UGT1A4 was low and unique because it was directed merely toward the 17-OH of both aglycones. UGT1A10 exhibited particularly high estradiol glucuronidation activity, the rate and affinity of which were significantly higher in the case of ␤-estradiol than with epiestradiol. UGT1A9 did not catalyze estradiol glucuronidation, but UGT1A9-catalyzed scopoletin glucuronidation was competitively inhibited by ␤-estradiol. UGT2B4, UGT2B7, and UGT2B17 exclusively conjugated the estradiols at the 17-OH position in a highly stereoselective fashion. UGT2B4 was specific for epiestradiol; UGT2B7 glucuronidated both diastereomers, with high affinity for epiestradiol, whereas UGT2B17 only glucuronidated ␤-estradiol. UGT2B15 glucuronidated both estradiols at the 3-OH, with a strong preference for epiestradiol. Human UGT2A1 and UGT2A2 glucuronidated both diastereoisomers at both hydroxyl groups. Microsomal studies revealed that human liver mainly yielded epiestradiol 17-O-glucuronide, and human intestine primarily yielded ␤-estradiol 3-O-glucuronide, whereas rat liver preferentially formed ␤-estradiol 17-O-glucuronide. Of the three recombinant rat UGTs that were examined in this study, rUGT2B1 was specific for the 17-OH of ␤-estradiol, rUGT2B2 did not catalyze estradiol glucuronidation, whereas rUGT2B3 exhibited high activity toward the 17-OH in both diastereoisomers. The results show that although many UGTs can catalyze estradiol glucuronidation, there are marked differences in their kinetics, regioselectivity, and stereoselectivity.

Summary In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

Nicotine biotransformation affects the smoking habits of addicted individuals and therefore their health risk. Using an improved analytical method, we have discovered that the human UDP-glucuronosyltransferase (UGT) 2B10, a liver enzyme previously unknown to conjugate nicotine or exhibit considerable activity toward any compound, plays a major role in nicotine inactivation by direct conjugation with glucuronic acid at the aromatic nitrogen atom. The K m value of recombinant UGT2B10 for nicotine (0.29 mM) was similar to that determined for human liver microsomes (0.33 mM), whereas the K m value of UGT1A4 for nicotine was almost 10-fold greater (2.4 mM). UGT2B10 was also more active than UGT1A4 in N-glucuronidation of cotinine (oxidative nicotine metabolite), whereas UGT2B7 exhibited only low nicotine glucuronidation activity and was essentially inactive toward cotinine. UGT1A9 did not glucuronidate nicotine or cotinine. Quantitative reverse transcription-polymerase chain reaction showed that UGT2B10 mRNA was exclusively expressed in human liver, whereas UGTs 1A4 and 2B7 were expressed at comparable, although somewhat lower, levels in liver and several other extrahepatic tissues, including kidney and intestine. These findings for UGT2B10 (but not for UGT1A4 and UGT2B7) were mirrored by human tissue activities because nicotine and cotinine glucuronidation rates in intestine microsomes were less than 0.1% that of human liver microsomes. These novel findings solve two seemingly separate questions: which UGT is primarily responsible for nicotine glucuronidation in human liver, and what conjugation reactions are catalyzed by UGT2B10.Nicotine is not carcinogenic by itself and might even have some beneficial therapeutic effects in some neurological diseases. Nonetheless, it is the major perpetrator of tobaccorelated diseases because nicotine addiction drives smokers to pursue the habit despite the known health hazards. Nicotine concentration in the blood increases sharply during cigarette smoking and then decreases rapidly because of metabolism and clearance, driving the addicted individual to reach for another cigarette. Hence, better understanding of nicotine metabolism can assist the development of treatments to reduce the health risks associated with nicotine addiction.Cytochrome P450 monooxygenase 2A6 (CYP2A6) catalyzes nicotine oxygenation and plays an important role in nicotine metabolism (Hukkanen et al., 2005;Nakajima and Yokoi, 2005). However, there is more to nicotine metabolism than CYP2A6 because both nicotine and its primary oxidation metabolite, cotinine, undergo direct N-glucuronidation at the aromatic nitrogen (Fig.

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