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.

This research suggests that the double covalent heme-protein linkage found in horse mitochondrial cytochrome c (cyt c) potentially plays a crucial role in the protein’s stability. The research compared the properties of a synthetically created heme peptide with a single covalent linkage to N-acetylmicroperoxidase-8, which mimics the natural cytochrome structure with double heme-protein linkages, and found the double-linkage model has greater stability and marked advantages.

Research Methodology

• The researchers compared the properties of N-acetylmicroperoxidase-8 (1), which contains residues 14-21 of horse mitochondrial cytochrome c (cyt c), to those of a synthetic mono-histidine coordinated heme peptide that contains a single covalent linkage (2).
• Through this comparison, the role of the double covalent heme-protein linkage in cytochrome c was examined.

Findings

• The research found that His-Fe(III) coordination is approximately 1.4 kcal/mol more favorable in the double covalent heme-protein structure (1) than in the single synthetic model (2). This means that model 1 has greater thermodynamic stability, which is a critical feature for protein function.
• Further EPR and resonance Raman studies revealed that both models 1 and 2 exhibit a thermal high-spin/low-spin ferric equilibrium, but model 1’s low-spin character is significantly more pronounced, implying greater stability.
• The research also found that a specific thioether, 2-(methylthio)ethanol (MTE), coordinates more strongly to model 1 than to model 2. However, it could only trigger the expected conversion of iron to the low-spin state, characteristic of ferric cyt c, in model 1.
• These findings suggest that the axial ligand field provided by an imidazole and a thioether is insufficient to induce a high-spin to low-spin conversion in a ferric porphyrin, which is addressed by the double covalent heme-protein linkage in cytochrome c.
• Moreover, the studies suggest that the double covalent heme-protein linkage in cytochrome c also potentially enables energetically favorable trans ligation of Met-80, which further increases the protein’s fold stability.

Proposition

• The researchers believe that a double covalent heme-protein linkage exists in cytochrome c as nature’s effective solution to the aforementioned limitations, enabling greater long-term protein stability.
• The stronger His-Fe(III) coordination enabled by these double linkages may serve to greatly enhance the effective ligand field strength of His-18, promoting the protein’s function.