Effect of Four Helix Bundle Topology on Heme Binding and Redox Properties

• Published in: Biochemistry (Easton) Vol. 37; no. 13; pp. 4635 - 4643
• Main Authors: Gibney, Brian R, Rabanal, Francesc, Reddy, Konda S, Dutton, P. Leslie
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 31.03.1998
• Subjects: Amino Acid Sequence; Carbon Monoxide - metabolism; Chromatography, Gel; Circular Dichroism; Guanidine; Heme - chemistry; Heme - metabolism; Hemeproteins - chemistry; Hemeproteins - metabolism; Iron Compounds - metabolism; Ligands; Molecular Sequence Data; Oxidation-Reduction; Peptides - chemical synthesis; Peptides - chemistry; Protein Binding; Protein Denaturation; Protein Structure, Secondary
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi971856s

We have designed two alternative four helix bundle protein scaffold topologies for maquette construction to examine the effect of helix orientation on the heme binding and redox properties of our prototype heme protein maquette, (α-SS-α)2, previously described as H10H24 [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A. J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425]. Conversion of the disulfide-bridged di-α-helical monomer of (α-SS-α)2 into a single polypeptide chain results in topological reorientation of the helix dipoles and side chains within a 62 amino acid helix-loop-helix monomer, (α-𝓁-α), which self-associates to form (α-𝓁-α)2. Addition of an N-terminal cysteine residue to (α-𝓁-α) with subsequent oxidation yields a 126 amino acid single molecule four helix bundle, (α-𝓁-α-SS-α-𝓁-α). Gel permeation chromatography demonstrated that (α-SS-α)2 and (α‘-SS-α‘)2, a uniquely structured variant of the prototype, as well as (α-𝓁-α)2 and (α‘-𝓁-α‘)2 assemble into distinct four helix bundles as designed, whereas (α-𝓁-α-SS-α-𝓁-α) elutes as a monomeric four α-helix bundle. Circular dichroism (CD) spectroscopy proves that these peptides are highly α-helical, and incorporation of four hemes has little effect on the helical content of the secondary structure. Four heme dissociation constants were evaluated by UV−visible spectroscopy and ranged from the 15 nM to 25 μM range for each of the peptides. The presence of Cotton effects in the visible CD illustrated that the hemes reside within the protein architecture. The equilibrium redox midpoint potentials (E m8) of the four bound hemes in each peptide are between −100 and −280 mV, as determined by redox potentiometry. The heme affinity and spectroelectrochemical properties of the hemes bound to (α-𝓁-α)2 and (α-𝓁-α-SS-α-𝓁-α) are similar to those of the prototype, (α-SS-α)2, and to bis-histidine ligated b-type cytochromes, regardless of the global architectural changes imposed by these topological rearrangements. The hydrophobic cores of these peptides support local electrostatic fields which result in nativelike heme chromophore properties (spectroscopy, elevated reduction potentials, heme−heme charge interaction, and reactivity with exogenous diatomics) illustrating the utility of these non-native peptides in the study of metalloproteins.