Structural consequences of b- to c-type heme conversion in oxidized Escherichia coli cytochrome b(562)

An NMR characterization of the 98Arg --> Cys variant of iron (III)-containi ng cytochrome b(562) from Escherichia coli has been performed and the solut ion structure obtained. This variant has a covalent bond between the heme a nd Cys 98, thus mimicking the heme binding in cytochrome c. The R98C cytoch rome is shown to have a significantly increased stability, compared to that of wild type, toward thermal and chemical denaturation. In water at 20 deg rees C it is 5.60 kT mol(-1) more stable than the WT protein, measured by e quilibrium guanidine hydrochloride denaturation. The structure has been obt ained through two-dimensional total correlation spectroscopy (TOCSY) and nu clear Overhauser effect spectroscopy (NOESY) experiments and through three- dimensional NOESY-N-15 heteronuclear multiple quantum coherence (HMQC). By these methods, 85% of protons and 100% of backbone nitrogens were assigned. 2145 meaningful nuclear Overhauser effects (NOEs) (20 NOEs per residue), 4 5 backbone (3)J values, and 397 pseudocontact shifts were used to obtain a family of 35 members, which were then energy-minimized. The root-mean-squar e deviation (RMSD) with respect to the average structure is 0.50 +/- 0.07 f or the backbone and 1.01 +/- 0.08 for the heavy atoms. The magnetic anisotr opy resulting from analysis of the pseudocontact shifts indicates an anisot ropy that is an intermediate between that of the wild-type, which is the sm allest, and cytochrome c. The g values confirm a higher anisotropy of the v ariant with respect to the wild-type protein. The chirality of the heme 2 a lpha carbon is the same as that in all naturally occurring cytochromes c. T he overall secondary structure and tertiary structure are very similar to t he wild type. The removal of Arg 98 causes a change in the pi-I-dependent p roperties. The pK(a), proposed to be due to deprotonation of the coordinate d histidine, is 1.5 units higher than in the wild type, consistent with the lack of the positive charge of Arg 98 close to the ionizable group. This i s further support for the coordinated histidine being the titratable group with an alkaline pK(a) in the wild-type protein. The pattern of the shifts of the heme methyl groups is different than in the wild-type protein, presu mably due to alteration of the electronic structure by the presence of the covalent bond between the protein and the heme. The difference in stability between the variant and wild-type protein is discussed in terms of the str uctural information.