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.

Ligand preference and orientation in b‐ and c‐type heme‐binding proteins

“…The simplest explanation is that certain heme ligation types have simply not yet been discovered; proteins with novel heme ligation motifs are still being found 60. Another reason is the difference in chemical properties of the possible ligands which influence the affinity and electrochemistry of the heme 19,20. For example, in the nonredundant database, b -type hemes rarely have His-Met ligation, while it is found in 16.4% of all c -type heme ligation.…”

“…To understand this difference in preference it is worth looking into the properties of Met heme ligation. Met binds more weakly to hemes than His especially in the oxidized heme form 20. Because of this, His-Met ligation will raise the heme E m by ≈150 mV relative to a bis-His heme in the same environment 30.…”

“…Each group has a different heme affinity,18,19 which will modify the resultant stability of the folded cofactor bound protein 20. In addition, each ligand will have a different affinity for the oxidized and reduced heme, which will shift the resultant in situ heme electrochemistry 20…”

Protein Design: Toward Functional Metalloenzymes

“…264 The construct containing two 3-methyl-histidines, [Δ H 3m] 2 , was used to examine the roles of the side chains on heme a , which differs from heme b by the presence of a C-2 hydroxyethylfarnesyl group and a C-8 formyl group. 265 The affinity, spectroscopy, and electrochemistry were compared between heme b and a heme a mimic, diacetyldeuterioporphyrin IX (DADPIX). While the binding of the ferrous forms of the two porphryins is the same at both sites in the construct, there is a marked difference in binding affinity in the ferric state for the construct containing DADPIX, which is attributed to the +160 mV (vs NHE) shift in redox potential.…”

“…Thus, the function of the electron-withdrawing groups on the porphyrin ring, such as the formyl group, is to raise the reduction potential by destabilizing the ferric form. 265 This work was extended by incorporating heme a itself. 892 The presence of the farnesyl tail on hemes a and o introduced hydrophobic interactions, which increased the binding affinity by 6.3 kcal mol −1 in both the ferrous and the ferric forms.…”