The solution structure of oxidized Escherichia coli cytochrome b562

The solution structure of the oxidized, paramagnetic form of cytochrome b(562) from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)J(HNH alpha) values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha 2 and alpha 3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b(562) of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha 4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pK(a) values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b(562) with the available structures of cytochromes c' which display a similar folding motif and similar pK(a) values but very little sequence similarity.