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 c(552) (Ht cyt c(552)) 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 (13)C nuclear magnetic resonance (NMR) chemical shifts for (13)CN(-) bound to heme iron trans to the axial His in Ht Fe(III) cyt c(552) variants. We observe a linear relationship between these (13)C 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 Hdelta1 and the backbone carbonyl of a Pro residue, we measured the pK(a) of the axial His Hdelta1 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 (13)Cbeta 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 (1)H 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 c(552) and display trends that correlate with E(m).
Bowman
Sarah E J
SE
Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Bren
Kara L
KL
D020816:Amino Acid Motifs; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D045304:Cytochromes c / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding
D006418:Heme; D006639:Histidine; D045304:Cytochromes c; D007501:Iron