CaltechTHESIS
  A Caltech Library Service

Wiring Inducible Nitric Oxide Synthase

Citation

Nguyen, Yen Hoang Le (2007) Wiring Inducible Nitric Oxide Synthase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fgsb-4m91. https://resolver.caltech.edu/CaltechETD:etd-09262006-134259

Abstract

The "wires project" in the Gray group has been focused on characterizing short-lived intermediates of Fe heme enzyme catalytic cycles by designing and synthesizing photosensitizers (wires) that bind to the protein active site with high affinity. The heart of this thesis is on rhenium channel binding and ruthenium surface binding wires for inducible nitric oxide synthase (iNOS). Binding and inhibition studies were conducted, electron transfer (ET) kinetics were studied, and iNOS catalytic activity was assayed for nitric oxide (NO) production.

Both channel and surface binding wires bind to iNOS with low micro molar affinity. Channel binding wires bind at the active site, closely interacting with the protoporphyrin IX iron heme (Fe heme). Characteristic spectral shifts of Fe heme perturbation were observed. The surface binding wires bind presumably at the hydrophobic patch of the oxygenase domain where the reductase domain was proposed to dock during electron transfer processes. The surface binding wire interacts closely with the Fe heme from the surface of the protein, but still close to where spectral shifts of the Fe heme were observed; however, the surface binding wire does not displace other channel binding wires, indicative of a second binding site.

Upon photo-excitation of all rhenium wires, the resting state Fe(III) heme is reduced to Fe(II) heme in less than 10 ns, characterized by transient absorption spectroscopy. This ET rate is orders of magnitude faster than Fe(III) reduction by the reductase domain (kET = 1 s-1) under biological conditions. In some cases, the wires were observed to ligate the Fe(II), creating a six-coordinate Fe(II) complex. The fully coordinated Fe(II) species is prevented from binding oxygen, and the catalytic mechanism is terminated. Another electron cannot be injected, and there is no production of NO. In the cases where the wire was shorter, ligation of the Fe(II) species was not observed. The Fe(II) remains five-coordinate, leaving room for oxygen to bind and for the mechanism to continue. In this case, NOS catalytic activity was assayed for the production of NO by photo-excitation of the wires. Complications of photodecomposition of NO indicators presented a challenge in data analysis. It is possible that a very small amount of NO was produced by photo-excitation of the wire; unfortunately, nothing definitely can be concluded. A new method to assay for NOS catalytic activity was proposed.

Both channel and surface binding wires led to many insights on substrate binding modes at the protein active site and on the surface; ET mechanisms were redefined, including amino acid radicals participating in electron transfer processes; and future directions for new wire were designed with hopes of accomplishing the long standing goal of characterizing high-valent Fe species.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:electron transfer; high-valent Fe heme; nitric oxide; nitric oxide synthase; wires
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Gray, Harry B.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Gray, Harry B.
  • Barton, Jacqueline K.
  • Winkler, Jay Richmond
  • Peters, Jonas C.
Defense Date:7 September 2006
Record Number:CaltechETD:etd-09262006-134259
Persistent URL:https://resolver.caltech.edu/CaltechETD:etd-09262006-134259
DOI:10.7907/fgsb-4m91
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3788
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:28 Sep 2006
Last Modified:16 Apr 2020 19:02

Thesis Files

[img]
Preview
PDF (YHN-Thesis.pdf) - Final Version
See Usage Policy.

8MB

Repository Staff Only: item control page