NMR analysis shows that a b-type variant of Hydrogenobacter thermophilus cytochrome c552 retains its native structure.

Conversion of Hydrogenobacter thermophilus cytochrome c(552) into a b-type cytochrome by mutagenesis of both heme-binding cysteines to alanines significantly reduces the stability of the protein (Tomlinson, E. J., and Ferguson, S. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5156-5160). To understand the effects of this change on the structure and dynamics of the protein, hetero-nuclear (15)N-edited NMR techniques have been used to characterize this b-type variant. The backbone (15)N, (1)H(N), and (1)H(alpha), and (1)H(beta) resonances of the protein have been assigned. Analysis of (3)J(HN)alpha coupling constants, nuclear Overhauser enhancement intensities, and chemical shift index data demonstrates that the four alpha-helices present in the wild-type protein are retained in the b-type variant. Comparison of the chemical shifts for the b-type and wild-type proteins indicates that the tertiary structures of the two proteins are closely similar. Some subtle differences are, however, observed for residues in the N-terminal region and in the vicinity of the heme-binding pocket. Hydrogen exchange studies show that there are 25 backbone amide protons that exchange very slowly in the b-type variant and confirm that the fluctuations within the b-type protein are of a similar extent to those in the wild-type protein. These data demonstrate the notable retention of the native secondary structure and tertiary fold despite the absence of covalent linkages between the heme group and the protein.