Functionalized de novo designed proteins: mechanism of proton coupling to oxidation/reduction in heme protein maquettes.

Proton exchange with aqueous media coupled to heme oxidation/reduction is commonly seen but not understood in natural cytochromes. Our synthetic tetrahelix bundle heme protein maquettes successfully reproduce natural proton coupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.4 to 10.3 in the maquette-associated acid/base group(s) upon heme reduction. Consequently, a 210 mV decrease in the heme redox potential is observed between the two extremes of pH. Potentiometry with resonance Raman and FTIR spectroscopy performed over a wide pH range strongly implicates glutamate side chains as the source of proton coupling below pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably, the pK values of several glutamates in the maquette are elevated from their solution value (4.4) to values as high as 7.0. It is suggested that these glutamates are recruited into the interior of the bundle as part of a structural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamate closest to the heme diminishes but does not abolish proton exchange. It is necessary to remove additional glutamates before pH-independent heme oxidation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the ferric heme and acid/base side chains. A similar mechanism is likely to exist in native redox proteins which undergo charge change upon cofactor oxidation/reduction.

Disruption of the heme iron-proximal histidine bond requires unfolding of deoxymyoglobin.

The unfolding behavior of 10 different distal heme pocket mutants of sperm whale deoxymyoglobin (deoxyMb) has been investigated. The effects of distal histidine (His 64) replacement were the primary focus; however, mutations at Leu 29, Val 68, and Ile 107 were also examined. Formation of the spectroscopically distinguishable heme intermediate (I') of deoxyMb was tracked as a function of pH and guanidinium chloride (GdmCl) concentration. The appearance of this intermediate signals cleavage of the iron-proximal histidine (His 93) bond. The key observations are as follows. (1) None of the distal heme pocket mutations investigated alter the nature of the heme intermediates that are formed under low pH unfolding conditions. (2) Unfolding of deoxyMb at high concentrations of GdmCl proceeds through the same heme intermediates that occur under low pH conditions. (3) The rate of the iron-histidine bond cleavage in an acidic medium is dramatically slowed when large hydrophobic residues (Leu and Phe) replace the distal histidine, whereas there is little correlation between the polarity of the residue at position 64 and the rate of denaturation by GdmCl. (4) However, apolar residues at position 64 enhance significantly the equilibrium resistance of deoxyMb to iron-histidine bond cleavage under both low pH and high GdmCl unfolding conditions. There is a direct correlation between the equilibrium pH and GdmCl values for maximum intermediate formation and the stabilities of the corresponding apoproteins. Collectively, these observations suggest that substantial unfolding of deoxyMb is required for Fe(II)-His 93 bond cleavage. Unlike the situation for aquometMb, heme loss from deoxyMb is not driven by protonation of the proximal histidine ligand. Instead, the process is mediated by more global unfolding of the protein that leads to solvation of the prosthetic group.

Structural and electronic properties of the heme cofactors in a multi-heme synthetic cytochrome.

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 interactions of PHs. The propensity to avoid such interactions could explain the inequivalent binding properties of the two hemes in the dimer. For the PHs protein wherein these these interactions cannot be mitigated by rotation of the heme, other rearrangements of the protein must occur. These rearrangements could force the second-bound heme to assume a high-spin configuration.