Author

Yue Dai

Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Clinical-Bioanalytical Chemistry

Department

Chemistry

First Advisor

Stuehr, Dennis

Subject Headings

Biochemistry

Abstract

Crystal structure of neuronal Nitric Oxide Synthase reductase (nNOSr) implies that large-scale domain motion is essential for electron transfer. However, the details are not well understood. To address this, we generated a functioning “Cys-lite” version of nNOSr and then replaced the nNOSr Glu816 and Arg1229 residues with Cys in the FMN and FAD domains (CL5SS) in order to allow cross-domain disulfide bond formation under pH 9 or to cross-linking using bis-maleimides. Cross-linked CL5SS exhibited a =95% decrease in cytochrome c reductase activity and reduction of the disulfide bond restored the activities. The results demonstrate that a conformational equilibrium involving FMN domains motion is essential for the electron transfer. A graded lengthening of the bis-maleimide cross-linkers was associated with an increase in activity, thus helping to define the distance constraints for domain opening. Stopped-flow kinetic studies showed cross-linking did not negatively affect the hydride transfer and interflavin electron but severally impaired the electron efflux from the FMN domain to its redox partner. How these findings impact our understanding of the nNOS catalytic cycle and details are discussed.

Staphylococcus aureus nitric oxide synthase (saNOS) helps S. aureus to maintain its antibiotics resistance, making saNOS a drug target. However, in vitro determination of saNOS inhibitor potency by activity assay is challenging because saNOS lacks an attached reductase. Herein, we employ the following approaches to optimize the in vitro assessment of NO synthesis by saNOS (1) B. subtillis flavodoxin YkuN and B. subtillis flavodoxin reductase FLDR were adopted as reductase partners for saNOS; (2) PEGylated-oxyhemoglobin was used for the direct capture of NO; (3) a 96-well plate format was used to increase the assay throughput. Our results showed that PEGylation of oxyHb minimizes the futile redox cycling within the flavoprotein and ensured effective electron transfer from produced NO to oxyHb. Nitric oxide produced by saNOS and cell cytosol was successfully detected by our assays. We also tested the inhibitory potency of six compounds derived from trimethoprim. They were confirmed to be H4F competitor with IC50 varying from 1 µM to 1 mM. The most potent inhibitor UCP111F26M is very specific to saNOS. Details of this inhibitor are discussed.

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