Variation and analysis of second-sphere interactions and axial histidinate character in c -type cytochromes

The electron-donating properties of the axial His ligand to heme iron in cytochromes c (cyts c) are found to be correlated with the midpoint reduction potential (E_m) in variants of Hydrogenobacter thermophilus cytochrome c₅₅₂ (Ht cyt c₅₅₂) in which mutations have been made in and near the Cys-X-X-Cys-His (CXXCH) heme-binding motif. To probe the strength of the His-Fe(III) interaction, we have measured ¹³C nuclear magnetic resonance (NMR) chemical shifts for ¹³CN^- bound to heme iron trans to the axial His in Ht Fe(III) cyt c₅₅₂ variants. We observe a linear relationship between these ¹³C chemical shifts and E_m, indicating that the His-Fe(III) bond strength correlates with E_m. To probe a conserved hydrogen bonding interaction between the axial His Hδ1 and the backbone carbonyl of a Pro residue, we measured the pK_a of the axial His Hδ1 proton (pK_a(2)), which we propose to relate to the His-Fe(III) interaction, reduction potential, and local electrostatic effects. The observed linear relationship between the axial His ¹³Cβ chemical shift and E_m is proposed to reflect histidinate (anionic) character of the ligand. A linear relationship also is seen between the average heme methyl ¹H chemical shift and E _m which may reflect variation in axial His electron-donating properties or in the ruffling distortion of the heme plane. In summary, chemical shifts of the axial His and exogenous CN^- bound trans to His are shown to be sensitive probes of the His-Fe(III) interaction in variants of Ht cyt c₅₅₂ and display trends that correlate with E_m.