Ammonia-oxidizing bacteria (AOB) generate energy by oxidizing ammonia (NH3) to nitrite (NO2-). After this process, nitrite-oxidizing bacteria convert NO2- into nitrate (NO3-). The complete oxidation of NH3 to NO3- by these two classes of microorganisms is known as the process of nitrification. AOB have also been shown to produce the greenhouse gas nitrous oxide (N2O) through a variety of enzymatic routes, the most recent of which was the enzyme cytochrome (cyt) P460, a soluble homodimeric enzyme containing one heme cofactor per subunit. Cyt P460 binds hydroxylamine (NH2OH) and oxidizes it by three electrons to produce the reactive {FeNO}6 intermediate, which reacts with a second equivalent of NH2OH to produce N2O. Recently, it was shown that a second-sphere glutamate (Glu) is necessary for the oxidation step, serving as a proton-relay. Cyt P460 is a c-type heme enzyme containing two thioether linkages germane to c-type cytochromes. However, this enzyme also uses a lysine (Lys) residue to form a third cross-link to the 13’ meso carbon of the heme. This cross-link alters the electronic structure of the heme and has been shown to be essential for catalysis. Mutation of the Lys residue produces UV-vis features reminiscent of standard cyt c’ and renders the enzyme incapable of NH2OH oxidation, despite the presence of the second-sphere Glu residue. The effect of the cross-link on enzyme activity has been unknown. When the cross-link is removed by mutation, the binding affinity for NH2OH does not substantially change compared to the wild type. The measured reduction potential becomes slightly more positive but does not change enough to explain differences in reactivity. Similarly, the electronic structure does not change in a catalytically meaningful way. Therefore, we sought to test if the cross-link serves a purely physical purpose in the enzyme. Herein, we report the first cross-link deficient crystal structure of cyt P460 from Nitrosomonas sp. AL212. This structure shows that the cross-link is essential to position the heme cofactor relative to the essential Glu residue to allow for electron transfer.

Effects of the heme-lysine cross-link on the cytochrome P460 active site

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.