STRUCTURAL AND ELECTRONIC-PROPERTIES OF THE HEME COFACTORS IN A MULTI-HEME SYNTHETIC CYTOCHROME

Resonance Raman; absorption, and electron paramagnetic resonance spect ra are reported for a water soluble, synthetic cytochrome. The protein is a variant of the cytochrome b maquette described by Robertson et a l. [Robertson, D. E., et al. (1995) Nature 368, 425-432] and is compos ed of 62 amino acid residues arranged in a di-alpha-helical unit which dimerizes in solution to form a four-helix bundle. Each di-alpha-heli cal unit contains histidine residues at the 10,10' positions which ser ve as ligands to the hemes. When protoheme IX is incorporated, both he mes in the dimer are bis-ligated and low spin. The two hemes are inequ ivalent with respect to both binding affinity and redox properties. To investigate the properties of the heme cofactors, spectroscopic studi es were conducted on peptides reconstituted with protoheme IX (PHa) an d several related variants. These hemes include 2-vinyldeuteroheme (2- VDH), 4-vinyldeuteroheme (4-VDH), protoheme III (PHs), and 1-methyl-2- oxomesoheme XIII (2-OMH). Collectively, the spectroscopic studies reve al the following: (1) 2-VDH, 4-VDH, and 2-OMH bind to the protein and form bis-ligated low-spin complexes similar to PHa. The structures of the two hemes in the dimers are identical as are the immediate protein environments around the bound cofactors. These results indicate that the redox inequivalence of the two hemes is due to heme-heme electroni c interactions rather than structural and/or environmental differences between the two cofactors. (2) The two hemes in the dimer are arrange d in a edge-to-edge arrangement wherein the oxo group (2-OMH) or the v inyl group(s) are in the hydrophobic interface between the two units w hich comprise the dimer. The propionic acid tails point outward toward the hydrophilic region and extend into the solvent. (3) The PHs prote in differs from the other synthetic proteins in that it contains one p entacoordinate, high-spin and one hexacoordinate, low-spin heme rather than two hexacoordinate low-spin cofactors. The open coordination sit e on the high-spin heme is inaccessible to exogenous imidazole but rea dily binds cyanide, suggesting that the alpha-helix containing the unb ound histidine is nearby and partially shields the coordination site. The high spin heme converts to low-spin at low-temperature, presumably via binding of the histidine residue on this nearby alpha-helix. It i s suggested that the different behavior observed for the PHs protein i s due to the fact that this heme is symmetric with respect to rotation about the alpha,gamma-axis of the macrocycle which bisects the meso-c arbons between the vinyl groups and propionic acid residues, This symm etry precludes rotational isomerism about the alpha,gamma-axis to esta blish an unhindered fit. In contrast, all the other hemes examined con tain at least one substituent smaller than a vinyl group which togethe r with the fact that two different alpha,gamma-rotational isomers are possible for each heme in the dimer could allow these hemes to avoid t he like-substituent-like-substituent heme-heme interactions of PI-Is. The propensity to avoid such interactions could explain the inequivale nt binding properties of the two hemes in the dimer. For the PHs prote in wherein these interactions cannot be mitigated by rotation of the h eme, other rearrangements of the protein must occur. These rearrangeme nts could force the second-bound heme to assume a high spin configurat ion.