No abstract

Proteins containing heme, iron(protoporphyrin IX) and its variants, continue to be one of the most-studied classes of biomolecules due to their diverse range of biological functions. The literature is abundant with reports of structural and functional characterization of individual heme proteins which demonstrate that heme protein reduction potential values, Em, span the range from –550 mV to +450 mV versus SHE. In order to unite these data for the purposes of global analysis, a new web-based resource of heme protein structure–function relationships is presented: the Heme Protein Database (HPD). This database is the first of its kind to combine heme protein structural classifications including protein fold, heme type and heme axial ligands, with heme protein reduction potential values in a web-searchable format. The HPD is located at http://heme.chem.columbia.edu/heme.php. The data illustrate that heme protein Em values are modulated over a 300 mV range by the type of global protein fold, a 600 mV range by the type of porphyrin and an 800 mV range by the axial ligands. Thus, the 1 V range observed in heme protein reduction potential values in biological systems arises from subtle combinations of these various factors.

The thermodynamics of ferric and ferrous haem affinity of a de novo designed four-alpha-helix bundle protein and the associated haem electrochemistry is described.

To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b-[H10A24]2 and heme b-[delta7-His]2, are four-alpha-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme-protein association, iron-histidine ligation, and heme-protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [Delta7-His]2 due to a stronger porphyrin-protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [delta7-His]2. The data indicate that porphyrin-protein hydrophobic interactions and heme iron coordination are responsible for the Kd value of 37 nM for the heme b-[delta7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin-protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited, <3.0 kcal/mol. The 1H+/1e- redox-Bohr effect of heme b-[H10A24]2 is due to the greater absolute stabilization of the ferric heme (4.1 kcal/mol) compared to the ferrous heme (1.4 kcal/mol) binding upon glutamic acid deprotonation, i.e., an electrostatic response mechanism. The 2H+/1e- redox-Bohr effect observed for heme b-[delta7-His]2 is due to histidine protonation and histidine dissociation of ferrous heme b upon reduction, i.e., a ligand loss mechanism. These results indicate that the contribution of porphyrin-protein hydrophobic interactions to heme affinity is critical to maintaining the heme bound in both oxidation states and eliciting an electrostatic response from these designed heme protein scaffolds.

The de novo design, synthesis, and characterization of a four-alpha-helix bundle scaffold containing heme ligated by 4-beta-(pyridyl)-l-alanine (Pal) is presented. The protein scaffold is highly helical, stable, and conformationally specific in the apo-state. Incorporation of heme using the designed bis-Pal axial ligation is shown using UV-visible and EPR spectroscopies. The observed heme midpoint reduction potential, +58 mV versus SHE, is 287 mV (6.8 kcal/mol) higher than the analogous bis-histidine-ligated heme protein.