Self-Assembly of Heme A and Heme B in a Designed Four-Helix Bundle: Implications for a Cytochrome c Oxidase Maquette

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Abstract
Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been introduced into two de novo designed four helix bundle proteins, [H10A24]2 and [H10H24]2, known to bind 2−4 equiv of heme B, respectively [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) Nature368, 425−432]. [H10A24]2, {[Ac-CGGGELWKL·HEELLKK·FEELLKL·AEERLKK·L-CONH2]2}2, binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10‘ positions with measured Kd values of Kd values of 50 and 800 nM). The heme A-protein complex, [heme A−H10A24]2, exhibits well-defined absorption spectra in both the ferric and ferrous states, and an electron paramagnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (Em8) was determined for [heme A−H10A24]2 at −45 mV (vs SHE), which is significantly higher than that of the protein bound heme B (−130 and −200 mV). The observation of a single midpoint redox potential for [heme A−H10A24]2 and a pair of midpoints for [heme B−H10A24]2 indicates that the di-α-helical monomers are oriented in an anti topology (disulfides on opposite sides of bundle) in the former (lacking heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the related [heme A−H10H24]2 which possess two distinct reduction potentials of +41 (31%) and −65 mV (69%). Self-assembly of the mixed cofactor {heme A−heme B−[H10A24]2} was accomplished by addition of a single equivalent of each heme A and heme B to [H10A24]2. The single midpoint redox potential of heme B, Em8 = −200 mV, together with the split midpoint redox potential of heme A in {heme A−heme B−[H10A24]2}, Em8 = +28 mV (33%) and −65 mV (67%), indicated the existence of both syn and anti topologies of the two di-α-helical monomers in this four helix bundle. Synthesis of the mixed cofactor [heme A−heme B−H10H24]2 was accomplished by addition of a 2 equiv of each heme A and heme B to [H10H24]2 and potentiometry indicated the pair of hemes B resided in the 10,10‘ sites and heme A occupied the 24,24‘ sites. The results indicate that heme peripheral structure controls the orientation of the di-α-helical monomers in the four-helix bundle which are interchangeable between syn and anti topologies. In the reduced form, [heme A−H10A24]2, reacts quantitatively to form [carbonmonoxy-heme A−H10A24]2 as evidenced by optical spectroscopy. The synthetic [heme A−H10A24]2 can be enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.