Nitric Oxide Reductase from Paracoccus denitrificans A Proton Transfer Pathway from the “Wrong” Side

Denitrification is an anaerobic process performed by several soil bacteria as an alternative to aerobic respiration. A key-step in denitrification (the N-Nbond is made) is catalyzed by nitric oxide reductase (NOR); 2NO + 2e + 2H → N2O + H2O. NOR from Paracoccus denitrificans is a member of the heme copper oxidase superfamily (HCuOs), where the mitochondrial cytochrome c oxidase is the classical example. It is situated in the cytoplasmic membrane and can, as a side reaction, catalyze the reduction of oxygen to water. NORs have properties that make them divergent members of the HCuOs; the reactions they catalyze are not electrogenic and they do not pump protons. They also have five strictly conserved glutamates in their catalytic subunit (NorB) that are not conserved in the ‘classical’ HCuOs. It has been asked whether the protons used in the reaction really come from the periplasm and if so how do the protons proceed through the protein into the catalytic site? In order to find out whether the protons are taken from the periplasm or the cytoplasm and in order to pinpoint the proton-route in NorB, we studied electronand proton transfer during a singleas well as multiple turnovers, using time resolved optical spectroscopy. Wild type NOR and several variants of the five conserved glutamates were investigated in their solubilised form or/and reconstituted into vesicles. The results demonstrate that protons needed for the reaction indeed are taken from the periplasm and that all but one of the conserved glutamates are crucial for the oxidative phase of the reaction that is limited by proton uptake to the active site. In this thesis it is proposed, using a model of NorB, that two of the glutamates are located at the entrance of the proton pathway which also contains two of the other glutamates close to the active site.