Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes.

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.

Spectroscopic characterization and assignment of reduction potentials in the tetraheme cytochrome C554 from Nitrosomonas europaea.

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is an essential electron transfer component in the biological oxidation of ammonia. The protein contains one 5-coordinate heme and three bis-His coordinated hemes in a 3D arrangement common to a newly characterized class of multiheme proteins. The ligand binding, electrochemical properties, and heme-heme interactions are investigated with Mössbauer and X- and Q-band (parallel/perpendicular mode) EPR spectroscopy. The results indicate that the 5-coordinate heme will not bind the common heme ligands, CN(-), F(-), CO, and NO in a wide pH range. Thus, cyt c(554) functions only in electron transfer. Analysis of a series of electrochemically poised and chemically reduced samples allows assignment of reduction potentials for heme 1 through 4 of +47, +47, -147, and -276 mV, respectively. The spectroscopic results indicate that the hemes are weakly exchange-coupled (J approximately -0.5 cm(-)(1)) in two separate pairs and in accordance with the structure: hemes 2/4 (high-spin/low-spin), hemes 1/3 (low-spin/low-spin). There is no evidence of exchange coupling between the pairs. A comparison of the reduction potentials between homologous hemes of cyt c(554) and other members of this new class of multiheme proteins is discussed. Heme 1 has a unique axial N(delta)-His coordination which contributes to a higher potential relative to the homologous hemes of hydroxylamine oxidoreductase (HAO) and the split-Soret cytochrome. Heme 2 is 300 mV more positive than heme 4 of HAO, which is attributed to hydroxide coordination to heme 4 of HAO.