The histidyl residues in ribonuclease-S. Photooxidation in solution and in single crystals; the iodination of histidine-12

Abstract

With molar ratios of methylene blue to ribonuclease-S of 1:30, histidyl residues 105 and 119 are destroyed by photooxidation at approximately equal rates. The loss of histidine 119 results in complete loss of enzymic activity; the loss of 105 is without effect on the activity. Histidine-12 in RNase-S is photooxidized at a slower rate than 105 or 119 and slower than the same residue in S-Peptide. Apparently histidine-12 is partially protected in the RNase-S complex. Loss of histidine-12 in S-Peptide results in loss of potential activity. Photooxidation of S-Peptide causes the oxidation of methionine-13 to the sulfoxide as well as the destruction of histidine-12. The sulfoxide can easily be reduced to yield a derivative where the only change is in histidine-12. Monoiodination of histidine-12 in S-Peptide on carbon-2(4) also resulted in complete loss of potential activity. Estimates of ratio of S-Protein to S-Peptide derivative association constants relative to that for the unmodified peptide show reductions in affinity by factors of about 700 for methionine-13 sulfone, 1900 for photooxidized histidine-12, 3200 for 2(4)-iodohistidine-12. The loss of histidine-119 markedly lowers the affinity of S-Protein for native S-Peptide. The photooxidation reaction was carried out in the solid state on single crystals of RNase-S. The qualitative effects were identical with those seen in solution, although the overall rate was slower. A comparison of X-ray diffraction patterns of native and photooxidized crystals of RNase-S showed only slight intensity changes. There was no evidence of any disordering of the crystal lattice. It is tentatively concluded that only small general conformational changes can have occurred, and that the activity effects can probably be attributed directly to the loss of the imidazole residues.