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Phototriggering nitric oxide synthas...

Keller, Gretchen Eva.

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Phototriggering nitric oxide synthase from Geobacillus stearothermophilus.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Phototriggering nitric oxide synthase from Geobacillus stearothermophilus./
作者:	Keller, Gretchen Eva.
面頁冊數:	171 p.
附註:	Source: Dissertation Abstracts International, Volume: 74-08(E), Section: B.
Contained By:	Dissertation Abstracts International74-08B(E).
標題:	Chemistry, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3560641
ISBN:	9781303068973

Phototriggering nitric oxide synthase from Geobacillus stearothermophilus.
Keller, Gretchen Eva.

Phototriggering nitric oxide synthase from Geobacillus stearothermophilus. - 171 p.

Source: Dissertation Abstracts International, Volume: 74-08(E), Section: B.

Thesis (Ph.D.)--California Institute of Technology, 2013.

Nitric oxide synthases (NOS) produce L-citrulline and nitric oxide (NO) in two turnovers from L-arginine via the bound intermediate, N-hydroxy-L-arginine. NO is an important biological signaling molecule, and NOS-like enzymes have been identified in all kingdoms of life. NOS enzymes utilize electrons from a reductase domain as well as a unique redox-active cofactor, tetrahydrobiopterin, in the activation of dioxygen. The NOS catalytic mechanism is only partially known, with proposed hihg-valent oxygenating intermediates similar to those of other monooxygenases. In the continued effort to understand the mechanism by which NOS catalytically produces NO, we have chosen to study NOS from the thermostable, nonpathogenic bacterium Geobacillus stearothermophilus (gsNOS).

ISBN: 9781303068973Subjects--Topical Terms:

1021807
Chemistry, General.

Phototriggering nitric oxide synthase from Geobacillus stearothermophilus.
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245 1 0 $a Phototriggering nitric oxide synthase from Geobacillus stearothermophilus.
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500 $a Source: Dissertation Abstracts International, Volume: 74-08(E), Section: B.
500 $a Adviser: Harry B. Gray.
502 $a Thesis (Ph.D.)--California Institute of Technology, 2013.
520 $a Nitric oxide synthases (NOS) produce L-citrulline and nitric oxide (NO) in two turnovers from L-arginine via the bound intermediate, N-hydroxy-L-arginine. NO is an important biological signaling molecule, and NOS-like enzymes have been identified in all kingdoms of life. NOS enzymes utilize electrons from a reductase domain as well as a unique redox-active cofactor, tetrahydrobiopterin, in the activation of dioxygen. The NOS catalytic mechanism is only partially known, with proposed hihg-valent oxygenating intermediates similar to those of other monooxygenases. In the continued effort to understand the mechanism by which NOS catalytically produces NO, we have chosen to study NOS from the thermostable, nonpathogenic bacterium Geobacillus stearothermophilus (gsNOS).
520 $a In this work, covalently modified gsNOS with a ruthenium(II) diimine photosensitizer has been prepared to circumvent the need for an external reductase domain, while maintaining access to the substrate-binding channel. In this way, we hoped to produce the NOS ferrous state by rapid electron injection and to be able to access intermediates that form downstream. Chapter two describes the engineered gsNOS for selective cysteine labeling with [Ru(bpy)2(IA-phen)] 2+ (IA-phen = 5-iodoacetamido-1,10-phenanthroline) to form the conjugate Ru(II)K115C-gsNOS. The labeled protein was characterized by mass spectrometry, UV-visible absorbance, and circular dichroism spectroscopy. Ru(II)K115C -gsNOS was crystallized and its structure solved to 2.6 A resolution. Chapter three details phototriggered reduction by laser flash-quench methodology using the reversible, reductive quencher p-methoxy-N,N-dimethylanaline. Transient absorption studies have shown that rapid phototriggered heme reduction occurs on the order of 625 ns. Binding of substrates and/or cofactor is known to alter the heme reduction potential in NOS. However, these observed reduction rates are independent of substrate/cofactor, suggesting that the system is driving force optimized. Studies performed in the presence of oxygen were complicated by side-reactions with the ruthenium label and small molecule quencher. Excitingly, enzymatic turnover was achieved by steady-state LED illumination at 470 nm using the irreversible reductive quencher diethyldithiocarbamate, and is described in chapter four. NO produced from light-driven Ru(II)K115C-gsNOS catalysis was measured by the Griess Assay and L-citrulline production confirmed by liquid chromatography-mass spectrometry. In chapter five, we show how phototriggered heme reduction can be used to monitor CO binding and potential studies of gas diffusion pathways, and substrate/cofactor effects are discussed. In all, the development of this ruthenium-modified gsNOS system has enabled preliminary studies of reduction kinetics, diatomic ligand binding, and light-driven catalysis and may provide a useful platform for further investigation into the catalytic cycle of NOS.
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