Structural and Electronic Properties of the Heme Cofactors in a Multi-Heme Synthetic Cytochrome

Abstract

Resonance Raman, absorption, and electron paramagnetic resonance spectra are reported for a water soluble, synthetic cytochrome. The protein is a variant of the cytochrome beta maquette described by Robertson et al. [Robertson, D. E., et al. (1995) Nature 368, 425-432] and is composed 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-helical unit contains histidine residues at the 10,10' positions which serve as ligands to the hemes. When protoheme IX is incorporated, both hemes in the dimer are bis-ligated and low spin. The two hemes are inequivalent with respect to both binding affinity and redox properties. To investigate the properties of the heme cofactors, spectroscopic studies were conducted on peptides reconstituted with protoheme IX (PHa) and 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 reveal 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 electronic interactions rather than structural and/or environmental differences between the two cofactors. (2) The two hemes in the dimer are arranged in a edge-to-edge arrangement wherein the oxo group (2-OMH) or the vinyl group(s) are in the hydrophobic interface between the two units which comprise the dimer. The propionic acid tails point outward toward the hydrophilic region and extend into the solvent. (3) The PHs protein differs from the other synthetic proteins in that it contains one pentacoordinate, high-spin and one hexacoordinate, low-spin heme rather than two hexacoordinate low-spin cofactors. The open coordination site on the high-spin heme is inaccessible to exogenous imidazole but readily bind cyanide, suggesting that the alpha-helix containing the unbound 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 is suggested that the different behavior observed for the PHs protein is 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-carbons between the vinyl groups and propionic acid residues. This symmetry precludes rotational isomerism about the alpha,gamma-axis to establish an unhindered fit. In contrast, all the other hemes examined contain at least one substituent smaller than a vinyl group which together with the fact that two different alpha,gamma-rotational isomers are possible for each heme in the dimer could allow these hemes to avoid the like-substituent--like-substituent heme--heme interac...

A heme-binding domain controls regulation of ATP-dependent potassium channels

“…The K ATP subunits Kir6.2 and SUR2A do not contain any CXXCH cytochrome c-like heme-binding motifs, as identified in the large conductance Ca 2+ -activated K + channel (BK channels) (12). However, SUR2A contains a CXXHX 16 16 H motif of the SUR2A subunit (residues 628-648) is located between the first transmembrane domain and the first nucleotide-binding domain (Fig. S1).…”

“…S1). The hypothesis that the CXXHX 16 H motif is involved in heme binding was therefore tested by mutagenesis (residues Cys628, His631, and His648).…”

“…S4 A, iv). The effect of mutations in the CXXHX 16 H motif (at C628S, H631A, and H648A) was also tested (Fig. 3 C-G).…”

“…To further quantify the interaction of heme with the SUR2A subunit, we examined heme binding to two model peptides and to a fragment of the SUR2A subunit (residues S615-L933) containing the entire CXXHX 16 H region. In assessing the spectroscopic properties of these heme-bound species, it is important to note that the spectra for regulatory heme proteins often differ from those of well-known heme proteins such as the globins and cytochrome c, most likely because the heme binds more weakly.…”

A differential scanning calorimetric study of the thermal unfolding of apo‐ and holo‐cytochrome b562

“…The fourhelix bundle is a very common structural motif, and the relative simplicity of the structure makes it an important target for de novo design efforts (DeGrado et al, 1989;Hahn et al, 1990;Hecht et al, 1990;Robertson et al, 1994;Bryson et al, 1995;Kalsbeck et al, 1996). Additionally, comparing apo-and holoprotein pairs provides valuable information regarding the importance of prosthetic groups to structure, stability, and assembly (Cocco & Lecomte, 1990;Moore et al, 1991;Feng et al, 1994).…”