title: Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette.
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) Nature 368, 425-432]. [H10A24](2), [Ac-CGGGELWKL x HEELLKK x FEELLKL x AEERLKK x L-CONH(2)](2)(2), binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10' positions with measured K(d) values of <0.1 and 5 nM, values 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 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 (E(m8)) 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-alpha-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, E(m8) = -200 mV, together with the split midpoint redox potential of heme A in heme A-heme B-[H10A24](2), E(m8) = +28 mV (33%) and -65 mV (67%), indicated the existence of both syn and anti topologies of the two di-alpha-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-alpha-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.
authors.affiliation: The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: B R
authors.initials: BR
authors.lastname: Gibney
authors.forename: Y
authors.initials: Y
authors.lastname: Isogai
authors.forename: F
authors.initials: F
authors.lastname: Rabanal
authors.forename: K S
authors.initials: KS
authors.lastname: Reddy
authors.forename: A M
authors.initials: AM
authors.lastname: Grosset
authors.forename: C C
authors.initials: CC
authors.lastname: Moser
authors.forename: P L
authors.initials: PL
authors.lastname: Dutton
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D011199:Potentiometry; D017433:Protein Structure, Secondary; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D010446:Peptide Fragments; D012996:Solutions; C027728:heme a; D006418:Heme; D003576:Electron Transport Complex IV