No abstract

Electron paramagnetic resonance (EPR) spectroscopy has long been a primary method for characterization of paramagnetic centers in materials and biological complexes. Transition metals in biological complexes have valence d-orbitals that largely define the chemistry of the metal centers. EPR spectra are distinctive for metal type, oxidation state, protein environment, substrates, and inhibitors. The study of many metal centers in proteins, enzymes, and biomimetic complexes has led to the development of a systematic methodology for quantitative interpretation of EPR spectra from a wide array of metal containing complexes. The methodology is now contained in the computer program SpinCount. SpinCount allows simulation of EPR spectra from any sample containing multiple species composed of one or two metals in any spin state. The simulations are quantitative, thus allowing determination of all species concentrations in a sample directly from spectra. This chapter will focus on applications to transition metals in biological systems using EPR spectra from multiple microwave frequencies and modes.

Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacterium Nitrosomonas europaea catalyzes the oxidation of NH(2)OH to HNO(2). The enzyme 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 other proteins. The spatial arrangement displays three types of diheme pairing motifs. At least four of the eight hemes are electronically coupled in two distinguishable pairs and one of these pairs is at the active site of the enzyme. Here, the use of quantitative simulation of the EPR signals allows determination of exchange couplings, and assignments of signals and reduction potentials to hemes of the crystal structure. The absence of any obvious heme-to-heme bonding pathway in the crystal structure suggests that the observed exchange interactions are derived from direct electronic overlap of porphyrin orbitals. This provides evidence for heme pairs which function as biological two-electron redox centers in electron-transfer processes.