title: Electron spin echo modulation and nuclear relaxation studies of staphylococcal nuclease and its metal-coordinating mutants.
abstract: Electron spin echo envelope modulation (ESEEM) spectroscopy has been applied to the determination of the number of water molecules coordinated to the metal in the binary complex of staphylococcal nuclease with Mn2+, to the ternary enzyme-Mn2+-3',5'-pdTp complex, and to ternary complexes of a number of mutant enzymes in which metal-binding ligands have been individually altered. Quantitation of coordinated water is based on ESEEM spectral comparisons of Mn2+-EDTA and Mn2+-DTPA, which differ by a single inner sphere water, and with Mn2+-(H2O)6. It was found that Mn2+ in the ternary complex of the wild-type enzyme has a single additional coordinated water, as compared to Mn2+ in the binary complex, confirming earlier findings based on T1 measurements of bound water [Serpersu, E. H., Shortle, D. L., & Mildvan, A. S. (1987) Biochemistry 26, 1289-1300]. Ternary complexes of the mutant proteins D40E, D40G, and D21Y have the same number of water ligands as the ternary complex of the wild-type enzyme, while the D21E mutant has one less water ligand. In order to maintain octahedral coordination geometry, these findings require two additional ligands to Mn2+ from the protein in the binary complex of the wild-type enzyme, probably Glu 43 and Asp 19, and one additional ligand from the protein in the ternary D40G and D21E complexes. Other ESEEM studies of ternary Mn2+ complexes of wild-type, D21E, and D21Y mutants indicate the coordination by Mn2+ of a phosphate of 3',5'-pdTp, as demonstrated by a 31P contact interaction of 3.9 +/- 0.3 MHz. Magnetic interaction of Mn2+ with 31P could not be demonstrated with the D40G and D40E mutants, suggesting that metal-phosphate distances are greater in these mutants than in the wild-type protein. In a parallel NMR study of the paramagnetic effects of enzyme-bound Co2+ (which occupies the Mn2+ site on the enzyme) on the T1 of 31P from enzyme-bound 3',5'-pdTp and 5'-TMP, it was found that metal to 5'-phosphate distances are 0.9-1.6 A shorter in ternary complexes of the wild-type enzyme and of the D21E mutant than in ternary complexes of the D40G mutant. In all cases, the 5'-phosphate of pdTp is in the inner coordination sphere of Co2+ and the 3'-phosphate is predominantly in the second coordination sphere.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: E H
authors.initials: EH
authors.lastname: Serpersu
authors.forename: J
authors.initials: J
authors.lastname: McCracken
authors.forename: J
authors.initials: J
authors.lastname: Peisach
authors.forename: A S
authors.initials: AS
authors.lastname: Mildvan
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D003035:Cobalt; D008345:Manganese; D008836:Micrococcal Nuclease; D002118:Calcium