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James Boswell (18th Century Biographer)

Biochemistry 35: 12241-12250 (1996)

Structural Investigations on the Coordination Environment of the Active-Site Copper Centers of Recombinant Bifunctional Peptidylglycine alpha-Amidating Enzyme

John S. Boswell, Brian J. Reedy, Raviraj Kulathila, David Merkler, and Ninian J. Blackburn

Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, P.O. Box 91000, Portland, Oregon 97291-1000, and Unigene Laboratories, 110 Little Falls Road, Fairfield, New Jersey 07004

Abstract

The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine R-amidating enzyme (R-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 ( 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2-3 histidines and 1-2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced enzyme data indicates that the non-histidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/Cl ligand per copper. The value of Ó(CO) (2093 cm -1 ) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the ¢Pro-PHM382s mutant M 314 I is totally inactive. Binding of the peptide substrate N-Ac-Tyr-Val- Gly causes minimum structural perturbation at the Cu(I) centers but appears to induce a more rigid conformation in the vicinity of the S-Met ligand. The unusually intense 8983 eV Cu K-absorption edge feature in reduced and substrate-bound-reduced enzymes is suggestive of a trigonal or digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to Cu I A and 2 histidines and 1 methionine as ligands to Cu I B. However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for CuA is also entirely consistent with the data.

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Biochemistry 34: 2857-2865 (1995)

The catalytic core of peptidylglycine alpha-hydroxylating monooxygenase: investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance.

B. A. Eipper, A. S. Quon, R. E. Mains, J. S. Boswell & N. J. Blackburn

Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97221.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that plays a key role in the biosynthesis of many peptides. Using site-directed mutagenesis, the catalytic core of PHM was found not to extend beyond Asp359. Shorter PHM proteins were eliminated intracellularly, suggesting that they failed to fold correctly. A set of mutant PHM proteins whose design was based on the structural and mechanistic similarities of PHM and dopamine beta-monooxygenase (D beta M) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in D beta M, to Phe79 altered the kinetic parameters of PHM. Disruption of either His-rich cluster contained within the PHM/D beta M homology domain eliminated activity, while deletion of a third His-rich cluster unique to PHM failed to affect activity; the catalytically inactive mutant PHM proteins still bound to a peptidylglycine substrate affinity resin. EPR and EXAFS studies of oxidized PHM indicate that the active site contains type 2 copper in a tetragonal environment; the copper is coordinated to two to three His and one to two additional O/N ligands, probably solvent, again supporting the structural homology of PHM and D beta M. Mutation of the Met residues common to PHM and D beta M to Ile identified Met314 as critical for catalytic activity.

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Carcinogenesis 12: 551-61 (1991)

Activation of chromium(VI) by thiols results in chromium(V) formation, chromium binding to DNA and altered DNA conformation.

K. M. Borges, J. S. Boswell, R. H. Liebross & K. E. Wetterhahn

Department of Biochemistry, Dartmouth College, Hanover, NH 03755.

The ability of the thiols glutathione, cysteine, beta-mercaptoethanol and dithiothreitol to effect chromium(VI)-induced DNA damage in vitro has been investigated. Reaction of pBR322 DNA with chromium(VI) in the presence of the thiols led to formation of chromium(V) and chromium--DNA adducts. The extent of chromium binding to DNA differed by several orders of magnitude among the thiols tested, in the order dithiothreitol greater than beta-mercaptoethanol much greater than cysteine greater than or equal to glutathione. The maximal level of chromium(V) formed also differed among the thiols tested, in the order beta-mercaptoethanol greater than dithiothreitol much greater than glutathione greater than or equal to cysteine. Electronic spectral studies of these reactions indicated that the rate of reduction of chromium(VI) is dependent on the thiol tested, in the order cysteine greater than dithiothreitol greater than glutathione greater than beta-mercaptoethanol. Electron paramagnetic resonance studies of these reactions indicate that a significant level of chromium(III) is detected only with cysteine. Chromium--DNA adducts formed by reaction of chromium(VI) in the presence of glutathione or cysteine did not lead to DNA conformational changes detectable upon agarose gel electrophoretic analysis. Changes in DNA conformation were detected as altered electrophoretic mobility of pBR322 DNA on agarose gels after reaction with chromium(VI) in the presence of dithiothreitol or beta-mercaptoethanol. Effects on DNA electrophoretic mobility, which depended on whether the initial conformation of the plasmid was linear or supercoiled, included altered and heterogeneous mobility, as well as complete inhibition of migration of the plasmid. Transmission electron microscopy of chromium--DNA complexes revealed aggregates of several plasmids, as well as condensation of individual plasmids into compact kinked forms. These effects may be due to cross-linking of DNA induced by chromium metabolites. These studies indicate that the levels of chromium bound to DNA are related to the levels and stabilities of the chromium(V) species formed upon reaction of chromium(VI) with the various thiols. Chromium--thiol interactions may play an important role in chromium(VI) genotoxicity.