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.
Abstract: 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.
Publication Date: 2004-02-11 PubMed ID: 14769043DOI: 10.1021/bi035531pGoogle Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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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.
Cite This Article
APA
Cowley AB, Lukat-Rodgers GS, Rodgers KR, Benson DR.
(2004).
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.
Biochemistry, 43(6), 1656-1666.
https://doi.org/10.1021/bi035531p Publication
Researcher Affiliations
- Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
MeSH Terms
- Animals
- Binding Sites
- Cytochromes c / chemistry
- Electron Spin Resonance Spectroscopy
- Enzyme Stability
- Ferric Compounds / chemistry
- Glycine / chemistry
- Heme / chemistry
- Hemeproteins / chemistry
- Histidine / chemistry
- Horses
- Imidazoles / chemistry
- Iron / chemistry
- Ligands
- Mercaptoethanol / analogs & derivatives
- Mercaptoethanol / chemistry
- Methionine / chemistry
- Peptide Fragments / chemistry
- Peptides / chemical synthesis
- Protein Structure, Secondary
- Spectrum Analysis, Raman
- Structure-Activity Relationship
Grant Funding
- P20 RR015566 / NCRR NIH HHS
Citations
This article has been cited 9 times.- Tanabe J, Nakano K, Hirata R, Himeno T, Ishimatsu R, Imato T, Okabe H, Matsuda N. Totally synthetic microperoxidase-11.. R Soc Open Sci 2018 May;5(5):172311.
- Zaidi S, Hassan MI, Islam A, Ahmad F. The role of key residues in structure, function, and stability of cytochrome-c.. Cell Mol Life Sci 2014 Jan;71(2):229-55.
- Nothnagel HJ, Preimesberger MR, Pond MP, Winer BY, Adney EM, Lecomte JT. Chemical reactivity of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination.. J Biol Inorg Chem 2011 Apr;16(4):539-52.
- Bowman SE, Bren KL. The chemistry and biochemistry of heme c: functional bases for covalent attachment.. Nat Prod Rep 2008 Dec;25(6):1118-30.
- Negron C, Fufezan C, Koder RL. Geometric constraints for porphyrin binding in helical protein binding sites.. Proteins 2009 Feb 1;74(2):400-16.
- Reedy CJ, Elvekrog MM, Gibney BR. Development of a heme protein structure-electrochemical function database.. Nucleic Acids Res 2008 Jan;36(Database issue):D307-13.
- Michel LV, Ye T, Bowman SE, Levin BD, Hahn MA, Russell BS, Elliott SJ, Bren KL. Heme attachment motif mobility tunes cytochrome c redox potential.. Biochemistry 2007 Oct 23;46(42):11753-60.
- Marboutin L, Boussac A, Berthomieu C. Redox infrared markers of the heme and axial ligands in microperoxidase: Bases for the analysis of c-type cytochromes.. J Biol Inorg Chem 2006 Oct;11(7):811-23.
- Di Paolo RE, Pereira PM, Gomes I, Valente FM, Pereira IA, Franco R. Resonance Raman fingerprinting of multiheme cytochromes from the cytochrome c3 family.. J Biol Inorg Chem 2006 Mar;11(2):217-24.
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