EFFECT OF 4 HELIX BUNDLE TOPOLOGY ON HEME-BINDING AND REDOX PROPERTIES

We have designed two alternative four helix bundle protein scaffold to pologies for maquette construction to examine the effect of helix orie ntation on the heme binding and redox properties of our prototype heme protein maquette, (alpha-SS-alpha)(2), previously described as H10H24 [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pid ikiti, R., Lear, J. D., Wand, A. J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425]. Conversion of the disulfide-bridged di-alpha -helical monomer of (alpha-SS-alpha)(2) into a single polypeptide chai n results in topological reorientation of the helix dipoles and side c hains within a 62 amino acid helix-loop-helix monomer, (alpha-l-alpha) which self-associates to form (alpha-l-alpha)(2). Addition of an N-te rminal cysteine residue to (alpha-l-alpha) with subsequent oxidation y ields a 126 amino acid single molecule four helix bundle, (alpha-l-alp ha-SS-alpha-l-alpha). Gel permeation chromatography demonstrated that (alpha-SS-alpha)(2) and (alpha'-SS-alpha')(2), a uniquely structured v ariant of the prototype, as well as (alpha-l-alpha)(2) and (alpha'-l-a lpha')(2) assemble into distinct four helix bundles as designed, where as (alpha-l-alpha-SS-alpha-l-alpha) elutes as a monomeric four alpha-h elix bundle. Circular dichroism (CD) spectroscopy proves that these pe ptides are highly alpha-helical, and incorporation of four hemes has l ittle effect on the helical content of the secondary structure. Four h eme dissociation constants were evaluated by UV-visible spectroscopy a nd ranged from the 15 nM to 25 mu M range for each of the peptides. Th e presence of Cotton effects in the visible CD illustrated that the he mes reside within the protein architecture. The equilibrium redox midp oint potentials (E-m?(8) of the four bound hemes in each peptide are b etween -100 and -280 mV, as determined by redox potentiometry. The hem e affinity and spectroelectrochemical properties of the hemes bound to (alpha-l-alpha)(2) and (alpha-l-alpha-SS-alpha-l-alpha) are similar t o those of the prototype, (alpha-SS-alpha)(2), and to bis-histidine li gated b-type cytochromes, regardless of the global architectural chang es imposed by these topological rearrangements. The hydrophobic cores of these peptides support local electrostatic fields which result in n ativelike heme chromophore properties (spectroscopy, elevated reductio n potentials, heme-heme charge interaction, and reactivity with exogen ous diatomics) illustrating the utility of these non-native peptides i n the study of metalloproteins.