Self-assembly of heme A and heme B in a designed four-helix bundle: Implications for a cytochrome c oxidase maquette

Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been i ntroduced into two de novo designed four helix bundle proteins, [H10A24](2) and [H10H24](2), known to bind 2-4 equiv of heme B, respectively [Robertso n, 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, 425-432]. [H10A24](2), {[Ac-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK.L-CONH2](2)} (2), binds two heme A molecules per four-helix unit via bis-histidine Ligat ion at the 10,10' positions with measured K-d values of <0.1 and 5 nM, valu es much lower than those measured for heme B (K-d values of 50 and 800 nM). The heme A-protein complex, [heme A-H10A24](2), exhibits well-defined abso rption spectra in both the ferric and ferrous states, and an electron param agnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (E-m8) was determined for [heme A-H 10A24](2) at -45 mV (vs SHE), which is significantly higher than that of th e protein bound heme B (-130 and -200 mV). The observation of a single midp oint redox potential for [heme A-H10A24](2) and a pair of midpoints for [he me B-H10A24](2) indicates that the di-or-helical monomers are oriented in a n anti topology (disulfides on opposite sides of bundle) in the former (lac king heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the r elated [heme A-H10H24](2) which possess two distinct reduction potentials o f +41 (31%) and -65 mV (69%). Self-assembly of the mixed cofactor Cheme A-h eme B-[H10A24](2)} was accomplished by addition of a single equivalent of e ach heme A and heme B to [H10A24](2). The single midpoint redox potential o f heme B, E-m8 = -200 mV, together with the split midpoint redox potential of heme A in theme A-heme B-[H10A24](2)}, E-m8 = +28 mV (33%) and -65 mV (6 7%), indicated the existence of both syn and anti topologies of the two di- cr-helical monomers in this four helix bundle. Synthesis of the mixed cofac tor [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 orientat ion of the di-alpha-helical monomers in the four-helix bundle which are int erchangeable between syn and anti topologies. In the reduced form, [heme A- H10A24](2), reacts quantitatively to form [carbonmonoxy-heme A-H10A24](2) a s evidenced by optical spectroscopy. The synthetic theme A-H10A24](2) can b e enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.