The structure of phosphate-free bovine ribonuclease A has been refined at 1.26-A resolution by a restrained least-squares procedure to a final R factor of 0.15. X-ray diffraction data were collected with an electronic position-sensitive detector. The final model consists of all atoms in the polypeptide chain including hydrogens, 188 water sites with full or partial occupancy, and a single molecule of 2-methyl-2-propanol. Thirteen side chains were modeled with two alternate conformations. Major changes to the active site include the addition of two waters in the phosphate-binding pocket, disordering of Gln-11, and tilting of the imidazole ring of His-119. The structure of the protein and of the associated solvent was extensively compared with three other high-resolution, refined structures of this enzyme.

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A complex of RNase A with a transition-state analog, uridine vanadate, has been studied by a combination of neutron and x-ray diffraction. The vanadium atom occupies the center of a distorted trigonal bipyramid, with the ribose oxygen 02' at the apical position. Contrary to expectations based on the straightforward interpretation of the known in-line mechanism of action of RNase, nitrogen NE2 of histidine-12 was found to form a hydrogen bond to the equatorial oxygen 08, while nitrogen NZ of lysine41 makes a clear hydrogen bond to the apical oxygen 02'. Nitrogen ND1 of histidine-119 appears to be within a hydrogenbond distance of the other apical oxygen, 07. Two other hydrogen bonds between the vanadate and the protein are made by nitrogen NE2 of glutamine-il and by the amide nitrogen of phenylalanine-120. The observed geometry of the complex may necessitate reinterpretation of the mechanism of action of RNase.Although a number of crystallographic investigations have elucidated the three-dimensional structure of RNase A (1-4) and its proteolytic modification RNase S (5-7), many details of the mechanism of action of this enzyme still remain to be conclusively determined. RNase hydrolyzes single-stranded RNA and requires a pyrimidine base on the 3' side of the cleaved phosphodiester linkage (Fig. 1). The stereochemistry of catalysis was investigated by Usher et aL (8,9), and these experiments have shown that both steps of the reaction proceed through an in-line mechanism. Although Usher did not identify the groups in the active site of the protein responsible for catalysis, these may be inferred from the published structures of RNase and of the complexes with inhibitors (6, 7, 10-12). In the first step of catalysis, histidine-12 removes a proton from the 2' oxygen of the pyrimidine ribose, making the oxygen nucleophilic and capable of attacking the phosphorus. In the transition state, the phosphorus becomes pentacoordinated, with the 2' and 5' oxygens assuming apical positions. The bond between the 5' oxygen and the ribose is cleaved, and the result of the first step of the reaction is a 2',3'-cyclic pyrimidine nucleotide. The second step of the reaction is initiated by an attack of a water molecule activated by histidine-119 on the phosphorus opposite the 2' oxygen, followed by formation of a pentacoordinated intermediate; finally, oxygen 02' is protonated by histidine-12, and a 3' nucleotide is formed. According to this interpretation, nitrogen NE2 of histidine-12 should be in the proximity of the 2' oxygen and nitrogen ND1 of histidine-119 should be near the 5' oxygen in order to fulfill their catalytic roles (13)(14)(15). In addition, because it has been known that lysine-41 is necessary for the catalysis and is present in the active site, this amino acid was usually placed adjacent to one of the other oxygens of the phosphate. Such a model was recently used in the design of an artificial enzyme, which showed limited RNase-like activity (16). Lindquist et al. (17) postulated that a complex of uridine ...

Cassette mutagenesis was used to exchange the suggested copper ligand Met121 in azurin to all other amino acids, and a stop codon. The mutant proteins were characterized by optical absorption spectroscopy and EPR. At low pH, all mutants exhibit the characteristics of a blue type 1 copper protein, indicating that methionine is not needed to create a blue copper site. At high pH, the Glu121 and the Lys121 mutants constitute a new form of protein-bound copper that is outside the range of type 1 copper.

The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.