SOLUTION STRUCTURE OF REDUCED CLOSTRIDIUM-PASTEURIANUM RUBREDOXIN

solution structure of reduced Clostridium pasteurianum rubredoxin (MW 6100) is reported here. The protein is highly paramagnetic, with iron( II) being in the S=2 spin state. The HP protons of the ligating cystei nes are barely observed, and not specifically assigned. Seventy-six pe rcent of the protons have been assigned and 1267 NOESY peaks (of which 1037 are meaningful) have been observed. Nonselective T-1 measurement s have been measured by recording four nonselective 180 degrees-tau-NO ESY at different tau values, and fitting the intensity recoveries to a n exponential recovery. Thirty-six metal-proton upper and lower distan ce constraints have been obtained from the above measurements. The use of such constraints is assessed with respect to spin delocalization o n the sulfur donor atoms. The solution structure obtained with the pro gram DYANA has been refined through restrained energy minimization. A final family of 20 conformers is obtained with no distance violations larger than 0.24 Angstrom, and RMSD values to the mean structure of 0. 58 and 1.03 Angstrom for backbone and all heavy atoms, respectively (m easured on residues 3-53), The structure is compared to the X-ray stru cture of the oxidized and of the zinc substituted protein, and to the available structures of other rubredoxins, In particular, the comparis on with the crystal structure and the solution structure of the Zn der ivative of the highly thermostable Pyrococcus furiosus rubredoxin sugg ested that the relatively low thermal stability of the clostridial rub redoxin may be tentatively ascribed to the loosening of its secondary structure elements. This research is a further achievement at the fron tier of solution structure determinations of paramagnetic proteins.