Correlations of structure and electronic properties from EPR spectroscopy of hydroxylamine oxidoreductase

Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacteriu m Nitrosomonas europaea catalyzes the oxidation of NH2OH to HNO2. The enzym e contains eight hemes per subunit which participate in catalytic function and electron transport. The structure of the enzyme shows a unique spatial arrangement of the eight hemes, subsets of which are now observed in four o ther proteins. The spatial arrangement displays three types of diheme pairi ng motifs. At least four of the eight hemes are electronically coupled in t wo distinguishable pairs and one of these pairs is at the active site of th e enzyme. Here, the use of quantitative simulation of the EPR signals allow s determination of exchange couplings, and assignments of signals and reduc tion potentials to hemes of the crystal structure. The absence of any obvio us heme-to-heme bonding pathway in the crystal structure suggests that the observed exchange interactions are derived from direct electronic overlap o f porphyrin orbitals. This provides evidence for heme pairs which function as biological two-electron redox centers in electron-transfer processes.