Spectroscopic Characterization and Assignment of Reduction Potentials in the Tetraheme Cytochrome c554fromNitrosomonasEuropaea

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Abstract
The tetraheme cytochrome c554 (cyt c554) 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 c554 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 ≈ −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 c554 and other members of this new class of multiheme proteins is discussed. Heme 1 has a unique axial Nδ-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.

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