The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.

Prostaglandin E 2 (PGE 2 ) is a key mediator in inflammatory response. The main source of inducible PGE 2 , microsomal PGE 2 synthase-1 (mPGES-1), has emerged as an interesting drug target for treatment of pain. To support inhibitor design, we have determined the crystal structure of human mPGES-1 to 1.2 Å resolution. The structure reveals three well-defined active site cavities within the membrane-spanning region in each monomer interface of the trimeric structure. An important determinant of the active site cavity is a small cytosolic domain inserted between transmembrane helices I and II. This extra domain is not observed in other structures of proteins within the MAPEG (MembraneAssociated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily but is likely to be present also in microsomal GST-1 based on sequence similarity. An unexpected feature of the structure is a 16-Å-deep cone-shaped cavity extending from the cytosolic side into the membrane-spanning region. We suggest a potential role for this cavity in substrate access. Based on the structure of the active site, we propose a catalytic mechanism in which serine 127 plays a key role. We have also determined the structure of mPGES-1 in complex with a glutathione-based analog, providing insight into mPGES-1 flexibility and potential for structure-based drug design.membrane protein | X-ray crystallography | enzyme mechanism P rostaglandins are potent lipid messengers and are involved in numerous homeostatic biological functions [for a review of eicosanoid biology, see review by C. D. Funk (1)]. They are enzymatically derived from the essential fatty acid arachidonic acid and the synthesis proceeds via the formation of prostaglandin H 2 (PGH 2 ), a reaction catalyzed by the constitutively active cyclooxygenase COX-1 and the inducible cyclooxygenase COX-2. PGH 2 acts as a substrate for a range of terminal prostaglandin synthases, including the PGE synthases (PGES, EC 5.3.99.3) that convert PGH 2 to PGE 2 .Microsomal prostaglandin E 2 synthase-1 (mPGES-1), colocalized and up-regulated in concert with COX-2, is the major source of inducible PGE 2 and is associated with inflammation and pain (2). Several studies support a role for mPGES-1 also in cancer cell proliferation and tumor growth (3). Because treatment with COX-2 selective inhibitors is associated with elevated cardiovascular risk, safer approaches involving, for example, PGE 2 reduction, are needed (4). Mice deficient in mPGES-1 have shown significantly reduced effect on hypertension, thrombosis, and myocardial damage compared with inhibition or disruption of COX-2, suggesting mPGES-1 to be a potential target for pharmaceutical intervention in various areas of diseases (2, 5).mPGES-1 belongs to a superfamily of Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism, the MAPEG family (6). Members of the MAPEG family can be found in prokaryotes and eukaryotes but not in archaea (7). The most closely related MAPEG member is the microsomal glutathione transferase-1...

We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa3) from Rhodobacter sphaeroides with dioxygen using the flow-flash methodology and compared the results to those obtained with the well-characterised bovine mitochondrial enzyme. The overall reaction sequence was the same in the two enzymes, but the extents and rates of the electron-transfer reactions differed, implying differences in redox potentials, and/or interaction energies between electrons and protons during oxygen reduction. As with the bovine enzyme, the R. sphaeroides enzyme displayed two major kinetic phases of proton uptake with rate constants of approximately 5000 s-1 and approximately 500 s-1 at pH 7.9, concomitant with the peroxy to oxoferryl and oxoferryl to oxidised states. The net number of protons taken up in the R. sphaeroides enzyme was about approximately 1.9, which implies that upon reduction, the enzyme has to pick up approximately 2.1 H+ from the medium. On the basis of the comparison of electron-transfer reactions in the two enzymes, we conclude that the transfer rate of the fourth electron to the binuclear centre is not only determined by the electron-transfer rate from haem a to the binuclear centre, but also by the electron equilibrium between CuA and haem a. In addition, in contrast to the bovine enzyme, where the electron- and proton-transfer rates during oxidation of the fully reduced enzyme by O2 are all faster than the overall turnover rate, in the R. sphaeroides enzyme, the slowest kinetic phase was rate limiting for the overall turnover. Moreover, the comparison of the reactions in the two systems shows that in the R. sphaeroides enzyme, the electrons are more evenly distributed among the redox centres during oxygen reduction. This enables investigations of effects also of minor perturbations on, e.g., the electron-transfer characteristics in mutant enzymes, for which this study forms the basis.

The rapid increase of antibiotic resistance has created an urgent need to develop novel antimicrobial agents. Here we describe the crystal structure of the promising bacterial target phospho-N-acetylmuramoyl-pentapeptide translocase (MraY) in complex with the nucleoside antibiotic tunicamycin. The structure not only reveals the mode of action of several related natural-product antibiotics but also gives an indication on the binding mode of the MraY UDP-MurNAc-pentapeptide and undecaprenyl-phosphate substrates.