title: The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site.
abstract: NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c(552) from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c(552) with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c(552) from the apo state on the addition of heme has been demonstrated using NMR techniques. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.
authors.affiliation: Oxford Centre for Molecular Sciences, Central Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QH, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Wain
authors.forename: T A
authors.initials: TA
authors.lastname: Pertinhez
authors.forename: E J
authors.initials: EJ
authors.lastname: Tomlinson
authors.forename: L
authors.initials: L
authors.lastname: Hong
authors.forename: C M
authors.initials: CM
authors.lastname: Dobson
authors.forename: S J
authors.initials: SJ
authors.lastname: Ferguson
authors.forename: L J
authors.initials: LJ
authors.lastname: Smith
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
chemical_list: D003574:Cytochrome c Group; C083951:cytochrome c553; D006418:Heme; C022302:cytochrome C-552