Insight into Heme Protein Redox Potential Control and Functional Aspects of Six-Coordinate Ligand-Sensing Heme Proteins from Studies of Synthetic Heme Peptides

We describe detailed studies of peptide-sandwiched mesohemes PSM^A and PSM^W, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSM^A with tryptophan (Trp) to give PSM^W generates additional energy via Trp side chain−porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His−Fe^III ligation to a greater extent than His−Fe^II ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (E_m,8 value). This is intriguing because converting PSM^A to PSM^W decreased heme solvent exposure, which would normally be expected to stabilize Fe^II relative to Fe^III. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b₅. Our studies of the PSMs also revealed that reduction of Fe^III to Fe^II reversed the relative affinities of the first and second His ligands for Fe (K₂^III > K₁^III; K₂^II < K₁^II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which Fe^II can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as “dynamic strain”, is that an exogenous ligand can compete with one of the His ligands in an Fe^II-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b₅. Exclusive binding of exogenous ligands by the Fe^II form of PSM^A led to positive shifts in its E_m,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.