Success in use of the enzyme nitrile hydratase (NHase) in commercial production of amides and other useful organic compounds has drawn much attention to the structure and function of this enzyme in recent years. NHase contains either a non-heme mononuclear Fe(III) site with N2S3O coordination or a non-corrin Co(III) site with similar set of donor atoms around it. Although the donor groups around iron (and cobalt) at the active site of the NHases have been identified by spectroscopic techniques and in one case by crystallography, the role of the M(III) center in the catalytic conversion of nitriles to amides has not been elucidated. In this proposed study, we plan to determine the intrinsic properties of the biological M(III) sites via studies on accurate models of the active sites of NHases. Unlike the few Fe(III) complexes reported as NHase models, our [MIIINxSyO] (M = Fe, Co) models will contain both carboxamido nitrogen and thiolato sulfur donors and a built-in labile site for further reactions at the metal centers. We will also modify one or more of the thiolato S centers to SO and SO2 group to accurately mimic the enzyme active site. We will study the pKa and reactivity of the water molecule at the M(III) site and variations in it with changes in the ligands around M(III). We will attempt to elucidate the mechanism of hydration of nitriles at such metal centers. We intend to find out whether (a) the nitriles first bind at the labile site and then are attacked by free water molecule (or hydroxide) or (b) the hydration of nitriles is catalyzed by the metal-bound hydroxide by a outer-sphere mechanism. In order to establish the mechanism of photoactivation of specific NHases by NO, we will study the reactions of the [FeIIINxSyO] with NO to determine whether the combination of carboxamido nitrogens and thiolato sulfurs allows binding of NO to the Fe(III) sites and whether such NO-adducts are photolabile. The reactivities of both S-containing and SO2 (or SO)-containing models will be compared to determine the effects of post-translational modification of the Cys-S residues in NHase. Unlike other non-heme iron centers in biology, the iron in NHases prefers to remain in the +3 oxidation state and promotes hydrolytic instead of oxidative reactions. We will perform electrochemical measurements to establish that ligation of negatively charged ligands and polarizable thiolato S atoms to Fe(III) provides extra stability of the +3 state of iron and makes the Fe-NHase quite unusual in comparison to other non-heme iron enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061636-03
Application #
6525923
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Preusch, Peter C
Project Start
2000-08-01
Project End
2004-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
3
Fiscal Year
2002
Total Cost
$173,658
Indirect Cost
Name
University of California Santa Cruz
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Halpenny, Genevieve M; Gandhi, Kavita R; Mascharak, Pradip K (2010) Eradication of Pathogenic Bacteria by Remote Delivery of Nitric Oxide via Light-Triggering of Nitrosyl-Containing Materials. ACS Med Chem Lett 1:180-183
Rose, Michael J; Mascharak, Pradip K (2008) Fiat Lux: selective delivery of high flux of nitric oxide (NO) to biological targets using photoactive metal nitrosyls. Curr Opin Chem Biol 12:238-44
Rose, Michael J; Mascharak, Pradip K (2008) Photoactive Ruthenium Nitrosyls: Effects of Light and Potential Application as NO Donors. Coord Chem Rev 252:2093-2114
Szundi, Istvan; Rose, Michael J; Sen, Indranil et al. (2006) A new approach for studying fast biological reactions involving nitric oxide: generation of NO using photolabile ruthenium and manganese NO donors. Photochem Photobiol 82:1377-84
Harrop, Todd C; Olmstead, Marilyn M; Mascharak, Pradip K (2004) Binding of CO to structural models of the bimetallic subunit at the A-cluster of acetyl coenzyme A synthase/CO dehydrogenase. Chem Commun (Camb) :1744-5
Patra, Apurba K; Rose, Michael J; Murphy, Karen A et al. (2004) Photolabile ruthenium nitrosyls with planar dicarboxamide tetradentate N(4) ligands: effects of in-plane and axial ligand strength on NO release. Inorg Chem 43:4487-95
Afshar, Raman K; Patra, Apurba K; Olmstead, Marilyn M et al. (2004) Syntheses, structures, and reactivities of (Fe-NO)6 nitrosyls derived from polypyridine-carboxamide ligands: photoactive NO-donors and reagents for S-nitrosylation of alkyl thiols. Inorg Chem 43:5736-43
Ghosh, Kaushik; Eroy-Reveles, Aura A; Avila, Belem et al. (2004) Reactions of NO with Mn(II) and Mn(III) centers coordinated to carboxamido nitrogen: synthesis of a manganese nitrosyl with photolabile NO. Inorg Chem 43:2988-97
Patra, Apurba K; Mascharak, Pradip K (2003) A ruthenium nitrosyl that rapidly delivers NO to proteins in aqueous solution upon short exposure to UV light. Inorg Chem 42:7363-5
Marlin, Dana S; Olmstead, Marilyn M; Mascharak, Pradip K (2003) Reaction of (mu-oxo)diiron(III) core with CO2 in N-methylimidazole: formation of mono(mu-carboxylato)(mu-oxo)diiron(III) complexes with N-methylimidazole as ligands. Inorg Chem 42:1681-7

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