INVESTIGATION OF THE STRUCTURE OF OXIDIZED PSEUDOMONAS-AERUGINOSA CYTOCHROME-C-551 BY NMR - COMPARISON OF OBSERVED PARAMAGNETIC SHIFTS AND CALCULATED PSEUDOCONTACT SHIFTS

Extensive main-chain and side-chain assignments are reported for the H-1 NMR spectrum of ferricytochrome c-551 from Pseudomonas aeruginosa at 323 K and pH 5.2. These were obtained by sequential assignments of two-dimensional scalar and dipolar correlation spectra. The low-spin (S = 1/2) ferric iron in the oxidized state gives rise to extensive contact and pseudocontact shifts for resonances in the ferricytochrome. Total redox-state-dependent shifts were computed by comparison to the previously assigned ferrocytochrome c-551. A set of 179 firmly assigned protons was selected that were expected to experience only pseudocontact shift contributions in the oxidized form. The pseudocontact shifts were calculated for the set by a standard model [Williams, G., Clayden, N. J., Moore, G. R., & Williams, R. J. P. (1985) J. Mol. Biol. 183, 447-460] using the atomic coordinates from the X-ray crystallographic determination of the oxidized form [Matsuura, Y., Takano, T., & Dickerson, R. E. (1982) J. Mol. Biol. 156, 389-409], and effective anisotropy and geometric factors were adjusted to minimize the sum of the squared differences between observed redox-state shifts and calculated pseudocontact shift contributions. The root-mean-squared deviation with the optimized parameters was 0.12 ppm. The optimized model was then used to calculate pseudocontact shifts for other assigned protons outside the basic set. The overall agreement and the lack of any systematic discrepancies provide evidence that there are no major structural differences among the solution and crystal conformations of the oxidized and reduced forms, within the inherent resolution of this computational approach. The computation proved to be a powerful tool for the stereospecific assignment of 70 additional spin systems in cyt c-551. The optimized anisotropy showed principal axes aligned approximately along the directions of Fe-pyrrole nitrogen and Fe-ligand vectors.