Enzyme hosts novel catalytic copper cluster The catalytic center of nitrous oxide re ductase displays a novel butterflyshaped cluster of four copper ions [Nat. Struct. Biol, 7,191 (2000)]. The discov ery was made when structural biologists Christian Cambillau and Kieron Brown at the National Center for Scientific Re search, Marseille, France, along with coworkers there and at New University of Lisbon, in Portugal, determined the crystal structure of the enzyme from Pseudomonas nautica. Nitrous oxide reductase reduces N 2 0 to N2 in the last step of the bacterial denitrification pathway (N0 3 " -> N0 2 " -> NO -> N 2 0 -^ N2). The pathway is intrinsic to organisms that use nitrogen oxides, rath er than oxygen, as electron acceptors for anaerobic respiration. Denitrification is important in agriculture and in reducing the level of N 2 0, a greenhouse gas, in the atmosphere. Nitrous oxide is "a very, very stable molecule compared with NO," Cambil lau points out. 'The lifetime for NO is counted in seconds, whereas the resi dence time for N 2 0 in the atmosphere is believed to be 150 years. Something special is needed to break this bond." Nitrous oxide reductases were known to be dimers and had been thought to contain four copper ions per monomer— two at each of two sites. One site, the CuA site, has spectroscopic and other similari ties to cytochrome oxidase and is the electron entry site. The other site, the Cuz site, was believed to be the catalytic site. Since N 2 0 is relatively unreactive, the type of copper center that could bind and reduce it is of particular interest, notes Amy C. Rosenzweig, an assistant professor of biochemistry at Northwest ern University, Evanston, Π1., who wrote an accompanying commentary. Cambillau and coworkers now find the Cuz site is a distorted tetrahedron made up of four copper ions coordinat ed to seven histidine residues and three hydroxide ions. The copper ions are ar ranged asymmetrically—the average distance between Cul and Cu3 or Cu4 is 3.10 A, whereas the average distance between Cu2 and Cu3 or Cu4 is 2.55 A. The shorter Cu-Cu distances could "suggest that Cu z may represent anoth er example of a metal-metal bond in na ture," Rosenzweig states. 'The finding of the butterfly-shaped tetracopper cluster geometry for Cuz is a
complete surprise," says William B. Tolman, a professor of chemistry at the Uni versity of Minnesota, Minneapolis. "Not only was it unforeseen by biochemists studying the properties of the enzyme, but it also is a new motif in the synthetic inorganic chemistry of copper." Exam ples of multicopper complexes exist, he notes, but the low coordination geome tries and short Cu-Cu distances "have not, to my knowledge, been seen before in a tri- or tetracopper cluster." The struc ture raises many new and exciting ques tions, he says, including the oxidation states of the copper ions and the nature of the metal-metal interactions. The team has shown that the CuA site is located in the monomer's carboxy ter minal domain and the Cuz site in its ami no terminal domain. In the dimer, the carboxy terminal domain of one monomer faces the amino terminal domain of the other, placing opposing CuA and Cu z sites within electron-transfer reach of
Ribbon diagram shows four copper ions (purple) surrounded by seven histidine residues (yellow) at catalytic site (Οΐχ) of nitrous oxide reductase. In the line structure, copper ions are coordinated to the histidines and three hydroxide ions.
each other. At the CuA site, reminiscent of cytochrome oxidase, two copper ions are bridged by two cysteines. 'The confir mation of the structure of the CuA site is gratifying insofar as it now puts the finish ing touches on what was a contentious de bate about the site," Tolman comments. The European researchers propose that catalysis involves N 2 0 binding to Cu4 where "there's room to bind and there's a channel from the outside," Cam billau notes. Electrons flowing from the CuA site would reduce the other three copper ions, enabling the Cu z site to serve as an electron reservoir for fast electron exchange, the team suggests. Mairin Brennan
Bristol-Myers Squibb loses Taxol patents A federal judge has ruled that many of Bristol-Myers Squibb's patent claims on Taxol, its popular cancer drug, are invalid, opening the door to generic competition. The decision shocked some industry observers. "It was a big surprise," says Hemant Shah, principal of HKS & Co., an independent research firm in Warren, NJ. 'We [the financial community] ex pected these matters to go to trial. Rarely does a judge issue summary judgments." Taxol, or paclitaxel, is Bristol-Myers' second largest product with 1999 sales of $1.5 billion, accounting for 5% of the com pany's total sales. The drug is approved by the Food & Drug Administration to treat breast cancer, Kaposi's sarcoma, nonsmall-cell lung cancer, and ovarian cancer. U.S. District Court Judge William Wall of Newark, N.J., granted several motions made byfivegeneric drug com panies seeking to invalidate U.S. patents 5670537 and 5641803, which cover the use of Taxol for treating breast cancer, lung cancer, and Kaposi's sarcoma. Wall did not overturn claims relating to ovari an cancer. The validity of those BristolMyers patent claims will be decided in a trial scheduled to begin May 1. The ruling is an ironic twist of fate for Bristol-Myers, which, in an effort to stave off generic competition, initiated the patent infringement case in 1997 against nine generic manufacturers. Some of the generic firms, however, spotting a hole in Bristol-Myers' case, fought back. "We maintained all along that Bris tol's patents were invalid," says Neil Flanzraich, vice chairman and president MARCH 6, 2000 C&EN
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