The oxidation of ammonia to NO2− by Nitrosomonas is not a straightforward process (see [13] for recent review). The first step is conversion of NH3 (or NH4+ cations) into NH2OH (Figure 14-2). This reaction requires molecular O2 plus two electrons, believed to be supplied by ubiquinol. The ammonia mono-oxygenase (AMO) responsible for this reaction has never been purified, but it has been identified by a suicide-inhibitor labelling method. From the sequences of the two subunits of the enzyme it is recognised to be very similar to a Cu-dependent methane mono-oxygenase for which a crystal structure has recently been obtained [14]. The structure of CH4-mono-oxygenase shows many transmembrane helices along with Cu atoms bound in more globular regions at the periplasmic side of the membrane. Unfortunately, the exact arrangement of the Cu ions at an active site is not clear but at least this structure of methane mono-oxygenase provides a framework for future understanding of the AMO. The idea that ubiquinol provides electrons for the AMO is supported by the fact that partially purified preparations of the enzyme can use duroquinol as electron donor.
Figure 14-2. A scheme for electron transport pathways in N. europaea. It is assumed that electrons derived from NH2OH are delivered from NH2OH dehydrogenase (Hao) to ubiquinone via cytochrome c554 and cytochrome cm which is a member of the NapC family (see text). It is assumed that cytochrome cm catalyses ubiquinol formation with concomitant uptake of H+ from the periplasmic side. The positioning of the active site of Amo on the periplasmic side is by analogy with methane mono-oxygenase. Protons released upon the putative oxidation of UQH2 by Amo are not shown but probably are released to the periplasm.
Assuming that ubiquinol is the source of electrons for ammonia mono-oxygenase, the mechanism for ubiquinone reduction must next be identified. This is thought to be linked to the oxidation of NH2OH to NO2−, a process that occurs in the periplasm. This is a four-electron reaction that is catalysed by a multi-heme protein of the c-type. The electrons are believed to pass on to a tetraheme cytochrome known a cytochrome c554. From there the path of the electrons is not firmly established. However, there is another tetraheme c-type cytochrome that is anchored to the membrane via a transmembrane helix and clearly belongs to the NapC/NirT group of proteins that are widely implicated in transferring electrons from ubiquinol or menaquinol to periplasmic reductases [15]; in one case, a respiratory NO2−-reductase from Wolinella succinogenes, there is experimental evidence for this. Given this precedent, and the similarity between the NH2OH oxidoreductase and the NO2−-reductase, it seems very likely that this NapC-type protein, sometimes called cytochrome cm552 in this context [13], catalyses electron transfer from cytochrome c554 to ubiquinone.
It seems that ubiquinol must be principally oxidised via two routes, at almost equal rates. Thus half of the ubiquinol molecules must be oxidised by the ammonia mono-oxygenase that, as explained above, requires two electrons for each molecule of ammonia converted into NH2OH. The other pathway of ubiquinol oxidation is through electron transfer to O2 with concomitant generation of the H+-electrochemical gradient. The exact pathway for this electron transfer has been uncertain but it is now thought likely to be via a cytochrome bc1 complex to an aa3-type oxidase. This view is based on information from the genome sequence, which shows the presence of the cytochrome bc1 complex as well as cytochrome aa3 as the sole oxidase [13]. Nitrosomonas europaea has two distinct loci for the expression of cytochrome c oxidases, which terminate the aerobic respiratory network. Both clusters contain the genes encoding the major catalytic subunit of the oxidase and a smaller subunit where electrons from cytochrome c enter the complex. Both copies of the small subunit have a stretch of amino acids diagnostic for binding of a dinuclear copper site (CuA). In that respect, both types of oxidase resemble the aa3-type cytochrome c oxidases found in mitochondria of eukaryotes and in many bacterial species. N. europaea does not have the potential to express a so-called cbb3-type oxidase, which is a uniquely bacterial type of oxidase with a relatively high affinity for O2 (Km of about 5 nM). Electron transport to O2 from NH2OH is strongly inhibited by classical inhibitors of the bc1 complex [16]. Until recently, the cytochrome bc1 was not recognised to be present in Nitrosomonas and so many schemes for electron flow have in the past been shown with direct electron transfer to the oxidase; this would give a lower H+ translocation stoichiometry and now seems unlikely.
A third route for oxidation of ubiquinol is via reversed electron transfer through a NADH-ubiquinone oxidoreductase complex, a process that generates the NAD(P)H. NADPH would be formed from NADH through the activity of a H+-motive-force-dependent transhydrogenase enzyme required for reductive biosynthesis. As explained in the introductory part of this chapter, this process is driven by the H+ motive force that is generated by electron transfer from ubiquinol to O2; both the cytochrome bc1 complex and cytochrome aa3 contribute to this generation. It has been estimated that about 5% of ubiquinol is oxidised by this reversed electron transfer pathway.