title: Evaluation of electron-withdrawing group effects on heme binding in designed proteins: implications for heme a in cytochrome c oxidase.
abstract: Heme a, the metalloporphyrin cofactor unique to cytochrome c oxidases, differs from the more common heme b by two chemical modifications, a C-2 hydroxyethylfarnesyl group and a C-8 formyl group. To elucidate a role of the C-8 formyl group, we compare the heme affinity, spectroscopy, and electrochemistry of a heme a mimic, Fe(diacetyldeuterioporphyrin IX) or Fe(DADPIX), with heme b, Fe(protoporphryrin IX) or Fe(PPIX), incorporated into a designed heme protein. The [Delta7-H3m]2 protein ligand, or maquette, selected for this study contains two equivalent bis-(3-methyl-L-histidine) heme binding sites within a four-alpha-helix bundle scaffold. The spectroscopic data on Fe(PPIX) and Fe(DADPIX) bound to [Delta7-H3m]2 demonstrate that these complexes are excellent synthetic analogues for natural cytochromes b and a, respectively. Comparison of the spectroscopic, electrochemical, and equilibrium thermodynamic data measured for the Fe(PPIX)-[Delta7-H3m]2 maquette with the previously reported Fe(PPIX)-[Delta7-His]2 complex demonstrates that changing the heme axial ligands to 3-methyl-L-histidine from L-histidine does not alter the resulting heme protein properties significantly in either oxidation state. Heme binding studies demonstrate that [Delta7-H3m]2 binds two ferrous Fe(DADPIX) or Fe(PPIX) moieties with similar dissociation constant values. However, in the ferric state, the data show that [Delta7-H3m]2 only binds a single Fe(DADPIX) and that one 2500-fold weaker than oxidized Fe(PPIX). The data demonstrate that the 4.6 kcal mol(-1) weakened affinity of [Delta7-H3m]2 for oxidized Fe(DADPIX) results in the majority of the 160 mV, 3.7 kcal mol(-1), positive shift in the heme reduction potential relative to Fe(PPIX). These data indicate that a role of the formyl group on heme a is to raise the iron reduction potential, thus making it a better electron acceptor, but that it does so by destabilizing the affinity of bis-imidazole sites for the ferric state.
authors.affiliation: Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, USA.
authors.forename: Jinyou
authors.initials: J
authors.lastname: Zhuang
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Amoroso
authors.forename: Ryan
authors.initials: R
authors.lastname: Kinloch
authors.forename: John H
authors.initials: JH
authors.lastname: Dawson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Baldwin
authors.forename: Brian R
authors.initials: BR
authors.lastname: Gibney
mesh_terms: D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004583:Electrons; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D007202:Indicators and Reagents; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011166:Porphyrins / Q000737:chemistry; D011199:Potentiometry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D007202:Indicators and Reagents; D010455:Peptides; D011166:Porphyrins; C027728:heme a; D006418:Heme; D003576:Electron Transport Complex IV