N-15 backbone dynamics of ferricytochrome b(562): Comparison with the reduced protein and the R98C variant

The backbone dynamics of ferricytochrome b(562), a four-helix bundle protei n from Escherichia coli, have been studied by NMR spectroscopy. The consequ ences of the introduction of a c-type thioether linkage between the heme an d protein and the reduction to the ferrous cytochrome have also been analyz ed. N-15 relaxation rates R-1 and R-2 and H-1-N-15 NOES were measured at pr oton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced pro tein as well as for the oxidized R98C variant. In the latter protein, an "a rtificial" thioether covalent bond has been introduced between the heme gro up and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffo ni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Bi ochemistry 39, 1499-1514]. The N-15 relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanose cond time scale were compared for the three species. The three forms are ra ther rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochro me b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S-2) are found f or residues belonging to loops 1 and 2. Higher mobility, as indicated by lo wer order parameters, is found for heme binding helices alpha1 and alpha4 i n the R98C variant with respect to the wild-type protein. The analysis requ ires a relatively long rotational correlation time (tau (m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular sha pe and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsec ond time scale has also been performed on oxidized wild-type and R98C cytoc hrome b(562). In a CPMG experiment, decay rates were analyzed in the presen ce of spin-echo pulse trains of variable spacing. The dynamic behavior on t his time scale is similar to that observed on the sub-nanosecond time scale , showing an increased mobility in the residues connected to the heme ligan ds in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity.