188 Biochemical Society Transactions (2006) Volume 34, part 1 Proton transfer in bacterial nitric oxide reductase U. Flock, J. Reimann and P. ¨ Adelroth 1 Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden Abstract The NOR (nitric oxide reductase) from Paracoccus denitrificans catalyses the two-electron reduction of NO to N 2 O (2NO + 2H + + 2e − → N 2 O + H 2 O). The NOR is a divergent member of the superfamily of haem- copper oxidases, oxygen-reducing enzymes which couple the reduction of oxygen with translocation of protons across the membrane. In contrast, reduction of NO catalysed by NOR is non-electrogenic which, since electrons are supplied from the periplasmic side of the membrane, implies that the protons needed for NO reduction are also taken from the periplasm. Thus NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site. The proton pathway has not been identified, and the mechanism and timing of proton transfer during NO reduction is unknown. To address these questions, we have studied the reaction between NOR and the chemically less reactive oxidant O 2 [Flock, Watmough and ¨ Adelroth (2005) Biochemistry 44, 10711–10719]. When fully reduced NOR reacts with O 2 , proton-coupled electron transfer occurs in a reaction that is rate-limited by internal proton transfer from a group with a pK a of 6.6. This group is presumably an amino acid residue close to the active site that acts as a proton donor also during NO reduction. The results are discussed in the framework of a structural model that identifies possible candidates for the proton donor as well as for the proton-transfer pathway. Introduction Bacterial NORs (nitric oxide reductases) are integral mem- brane proteins that catalyse the reduction of NO to N 2 O (eqn 1) as part of the denitrification process: 2NO + 2H + + 2e − → N 2 O + H 2 O (1) The largest subunit in the NORs was shown by sequence similarity to be a divergent member of the superfamily of O 2 -reducing HCuOs (haem-copper oxidases), which are characterized by having a catalytic subunit with six invariant histidine residues at the same predicted positions in 12 trans- membrane helices [1,2]. Two of these histidine residues co- ordinate a low-spin haem, one co-ordinates a high-spin haem and the remaining three histidine residues co-ordinate a copper ion (hence the name), which in the NORs is replaced by a non-haem iron [3,4]. The purified NOR from Paracoccus denitrificans contains two subunits: NorB and NorC. NorB is the catalytic subunit harbouring the low-spin haem b, the high-spin haem b 3 , and the non-haem iron, Fe B , where the latter two form a bi- nuclear centre which is the site of NO reduction. NorC is a membrane-anchored protein harbouring a haem c, and is presumably the site of electron entry from the water-soluble electron donor. In the HCuOs, the reduction of oxygen to water (eqn 2) is coupled with the generation of an electrochemical proton gradient across the membrane. This is done by exclusively using protons from the ‘inside’ (bacterial cytoplasm) for water Key words: electron transfer, haem-copper oxidase, nitric oxide reductase, Paracoccus denitrificans, proton-transfer pathway. Abbreviations used: HCuO, haem-copper oxidase; NOR, nitric oxide reductase. 1 To whom correspondence should be addressed (email piaa@dbb.su.se). formation, and in addition, for each electron transferred to oxygen, one proton is pumped across the membrane (eqn 2). O 2 + 8H + in + 4e − → 2H 2 O + 4H + out (2) In contrast, available data indicate that NO reduction by NOR is non-electrogenic [5,6], i.e. not coupled with charge translocation across the membrane, despite the free energy available from NO reduction (E  0 =+1.2 V) being even larger than that from O 2 reduction (E  0 =+0.8 V). As electrons are supplied by soluble donors from the periplasmic side of the membrane, the lack of electrogenicity in NOR implies that the protons needed for NO reduction (see eqn 1) are also taken from the periplasm. This means that NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site, which is buried in the lipid bilayer. In order to study the mechanism of proton transfer into the active site in NOR, we have used the capability of NOR to use O 2 instead of NO as the oxidant [7,8] in combination with the ‘flow-flash’ technique [9]. Because of the chemical proton- coupled side-reactions of NO in water [6], O 2 is a more suitable oxidant for direct measurements of proton uptake. In the flow-flash technique [10], fully reduced CO-bound NOR is mixed rapidly with an oxygenated solution. Then, a short laser flash breaks the haem b 3 Fe–CO bond and the binding of dioxygen and its subsequent stepwise reduction by electron and proton transfer into the active site is followed by time-resolved optical spectroscopy. Our results identified an internal group in the vicinity of the active site that acts as a proton shuttle during catalysis. In this review, possible candidates for this group and a putative proton-transfer pathway from the periplasm into the active site are discussed in the framework of a structural model. C  2006 Biochemical Society