A possible role for the covalent heme-protein linkage in cytochrome c revealed via comparison of N-acetylmicroperoxidase-8 and a synthetic, monohistidine-coordinated heme peptide.
N-Acetylmicroperoxidase-8 (1) contains heme and residues 14-21 of horse mitochondrial cytochrome c (cyt c). The two thioether bonds linking protein to heme in cyt c are present in 1, and the native axial ligand His-18 remains coordinated to iron. As an approach to probing structural or functional roles played by the double covalent heme-protein linkage in cyt c, we have initiated a study in which the properties of 1 are compared with those of a synthetic mono-His coordinated heme peptide containing a single covalent linkage (2). One consequence of the greater conformational restriction imposed on peptide conformation in 1 is that His-Fe(III) coordination is approximately 1.4 kcal/mol more favorable in 1 than in 2. This highlights a clear advantage conferred to cyt c by having two covalent heme-protein linkages rather than one: greater thermodynamic stability of the protein fold. EPR (11 K) and resonance Raman (298 K) studies reveal that 1 and 2 exhibit a thermal high-spin/low-spin ferric equilibrium but that low-spin character is considerably more pronounced in 1. In addition, the thioether 2-(methylthio)ethanol (MTE) coordinates 0.5 kcal/mol more strongly to 1 than to 2 in 60:40 H(2)O/CH(3)OH and only triggers the expected conversion of iron to the low-spin state characteristic of ferric cyt c in the case of 1. This demonstrates that the axial ligand field provided by an imidazole and a thioether is too weak to induce a high-spin to low-spin conversion in a ferric porphyrin. Our results suggest that a conformationally constrained double covalent heme-protein linkage, as exists in 1 and its parent protein cyt c, is an effective solution that nature has evolved to circumvent this limitation. We propose that the stronger His-Fe(III) coordination enabled by such a linkage serves to markedly enhance the effective ligand field strength of His-18. Our studies with 1 and 2 suggest that a double covalent linkage in cyt c may also enable energetically more favorable trans ligation of Met-80 than would be possible if only a single linkage were present. This would serve to further increase the stability of the protein fold and perhaps to increase the effective ligand field strength of Met-80 as well.
Cowley
Aaron B
AB
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Lukat-Rodgers
Gudrun S
GS
Rodgers
Kenton R
KR
Benson
David R
DR
D000818:Animals; D001665:Binding Sites; D045304:Cytochromes c / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005290:Ferric Compounds / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006736:Horses; D007093:Imidazoles / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008623:Mercaptoethanol / Q000031:analogs & derivatives* / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis*; D017433:Protein Structure, Secondary; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship
D005290:Ferric Compounds; D006420:Hemeproteins; D007093:Imidazoles; D008024:Ligands; D010446:Peptide Fragments; D010455:Peptides; D006418:Heme; D006639:Histidine; C003859:methylthioethanol; D008623:Mercaptoethanol; C029899:imidazole; D045304:Cytochromes c; D008715:Methionine; D007501:Iron; C071067:N(alpha)-acetylmicroperoxidase-8; D005998:Glycine