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  • Open Daily: 10am - 10pm
    Alley-side Pickup: 10am - 7pm

    3038 Hennepin Ave Minneapolis, MN
    612-822-4611

Open Daily: 10am - 10pm | Alley-side Pickup: 10am - 7pm
3038 Hennepin Ave Minneapolis, MN
612-822-4611
The Chemistry of the Heme Domain of Nitric Oxide Synthase: Toward a Detailed Mechanism of N-G -Hydroxy-L-Arginine Oxidation.

The Chemistry of the Heme Domain of Nitric Oxide Synthase: Toward a Detailed Mechanism of N-G -Hydroxy-L-Arginine Oxidation.

Woodward, Joshua John

Paperback

Chemistry

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ISBN10: 1243668482
ISBN13: 9781243668486
Publisher: Proquest Umi Dissertation Pub
Pages: 162
Weight: 0.66
Height: 0.35 Width: 7.44 Depth: 9.69
Language: English
Nitric oxide synthase (NOS) is a P450 mono-oxygenase that catalyzes the oxidation of L-arginine to citrulline and NO through the stable intermediate NG-hydroxy-L-arginine (NHA). The oxidation of NHA is a unique reaction catalyzed by NOS. Currently, there is little direct evidence in support of the nature of the heme bound oxidant (i.e. ferric-peroxo vs. FeIV=O(por-+)) responsible for this transformation or the role of the pterin cofactor involved in this reaction. To address these questions we have developed a new method for incorporating unnatural porphyrins in the heme domain construct of the inducible nitric oxide synthase. This method is generally applicable to all heme proteins that can be expressed recombinantly in E. coli. Using these heme-substituted proteins, mutated NOS, and a substrate analog we have characterized a model reaction of NOS, known as the peroxide shunt. By correlating product distributions observed in this reaction with peroxidase activity, strong support for the involvement of a ferric-peroxo intermediate in the second step of NOS is presented. Further, the use of manganese substituted NOS provides support of HNO as the initial inorganic product generated during the peroxide shunt. The role of the pterin-centered radical has been probed using both the peroxide shunt and a chemical donor of HNO. These results show that NO production by the peroxide shunt is dependent on the presence of a pterin radical and provide that HMO may act as a the intermediate that is responsible for reducing the pterin radical in during NHA oxidation. These observations provide evidence in support of HNO as an intermediate on the path to NO production. Finally, a series of substrate analogs is characterized to address the role of water and active site hydrogen bonding in the activation of oxygen by NOS. A detailed understanding of these analogs will potentially provide insight into the mechanism by which NOS is able to utilize two separate heme-bound oxidants during each of the two half reactions involved in catalyzing nitric oxide.

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