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 In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A).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.
Highlights
There has been much interest in characterizing the structural and dynamical properties of the apo forms of heme-binding proteins to gain insight into the stability, folding, and assembly of these ubiquitous proteins
It has been proposed that holo H. thermophilus cytochrome c552 is generated spontaneously, implying that the apo state of this protein has sufficient structure to form a binding site for heme, the occupancy of which could be followed by covalent bond formation [17]
Further insight into the spontaneous formation of holo H. thermophilus cytochrome c552 has come from the finding that replacement of the two cysteine residues of the CXXCH motif by alanines (C11A/C14A) results in a protein that binds heme noncovalently, i.e. in the formation of a cytochrome b variant of the cytochrome c552 [19]. This protein is the only example of a conversion of a c-type to a b-type cytochrome through mutation and raises questions about the acquisition of the cytochrome c fold since the cytochrome b variant appears to have essentially the same native
Summary
There has been much interest in characterizing the structural and dynamical properties of the apo forms of heme-binding proteins to gain insight into the stability, folding, and assembly of these ubiquitous proteins (e.g. see Refs. 1–3). These results suggest that the bound ANS is located in the heme binding pocket, which would be at least partially formed in the apo state.
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