A high-resolution NMR study of long-lived water molecules in both oxidation states of a minimal cytochrome c.

The interaction of water with oxidized and reduced cytochrome c from the Gram-positive bacterium Bacillus pasteurii (a 71-amino acid long monoheme cytochrome) is investigated through CLEANEX experiments and N-15-edited ePHOGSY and Tr-ROESY experiments. It appears that a water molecule gives rise to dipolar cross-relaxation with the amide protons of Gly74 and Ile75, with a residence time longer than 0.4 ns, to account for a negative NOE. Such water molecule is present in both the oxidized and reduced species and in the X-ray structure. It appears to have a structural role. Other possible roles are discussed by comparison with the water molecules present in other c-type cytochromes. The amide proton of Cys35 is found to exchange rapidly with the solvent in the oxidized but not in the reduced protein, at variance with H/D exchange experiments, which probe a different time scale. The present data confirm that electron-transfer proteins evolved to minimize reorganization energy upon change of the oxidation state, even though the consequent variation of charge of the metal ion may induce some changes in the structure and/or dynamics of the protein.