Proton NMR study of chemically modified horse heart ferricytochrome c confirms the presence of histidine and lysine-ligated conformers in 30% acetonitrile solution

Comparison of the 1H NMR spectra for guanidinated ferricyt c and chloro(terpyridine)platinum(II)-modified ferricyt c in 30% acetonitrile (ACN) solution with that for ferricyt c in 30% ACN is reported. The absence of the heme methyl proton resonances characteristic of the IV*-form (Lys-ligated) in the NMR spectrum of guanidinated ferricyt c in 30% ACN solution confirms that a lysine-ligated form of ferricyt c is produced in 30% ACN solution. The absence of the 8-methyl heme proton resonance of the V*-form in the NMR spectrum of chloro(terpyridine)platinum(II)-modified ferricyt c in 30% ACN solution demonstrates that a bis-His-ligated form of ferricyt c is produced in 30% ACN, not a hydroxide ligated form, as previously proposed. The revised assignment for the V* form of ferricyt c in mixed media explains differences between the exchange network we previously reported for ferricyt c in 30% ACN [Protein Sci. 10 (2001) 2291] as versus that reported by Dopner et al. at high pH [J. Am. Chem. Soc. 120 (1998) 11246]. Lys- and His-ligated forms are known to be produced in the presence of denaturants in protein folding studies of ferricyt c. Consequently, the exchange network between these non-native forms of ferricyt c in 30% ACN may have biological relevance for ferricyt c folding.