Date

December 2009

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Science & Engineering

Institution

Oregon Health & Science University

Abstract

Our research goal is to understand the mechanisms by which metalloproteins sense and detoxify nitric oxide (NO). We have characterized two different bacterial sensor proteins which employ different metal cofactors to detect NO. Further, we have investigated the NO dioxygenase reaction utilized by the hemoglobin homologues of both mammals and bacteria to detoxify NO. These reactions are important for understanding how NO levels are determined and controlled in both mammalian and bacterial systems. DevS is a heme-based sensor kinase from Mycobacterium tuberculosis (MTB) which senses CO, NO and hypoxia. Upon reception of these signals, DevS autophosphorylates and transfers the phosphate group to the response regulator DevR, which induces a regulon associated with entrance of the bacterium into a dormant state. We use UV-vis and resonance Raman (RR) spectroscopy of full-length and truncated proteins as well as activity assays of wild type (wt) and variant forms in Fe(II), CO, NO, and O[subscript 2]-bound states to determine the mechanism by which DevS differentiates exogenous ligands and communicates this information from the heme to the kinase domain. Our results identify a specific hydrogen bond network, stabilized by distal residue Tyr171, which is required to differentiate between the activating ligands CO and NO and the inhibitory ligand O[subscript 2]. This signal is communicated to the kinase domain by interactions between the heme-binding GAF-A domain and second GAF domain called GAF-B. We have also investigated the NO-sensing transcriptional repressor NsrR from Bacillus subtilis. NO relieves repression of flavohemoglobin and nitrite reductase genes by NsrR. We use UV-vis, EPR, and RR spectroscopy to determine that this protein contains a [4Fe-4S] which is reactive toward NO. Thus, it seems likely that conformational changes caused by the binding of NO to the Fe-S cluster of NsrR are responsible for its NO-sensing ability. Finally, we investigated the NO dioxygenase mechanism of myoglobin. This reaction represents a major route of NO detoxification for both mammals and bacteria. NO reacts with the oxy-heme, leading to complete conversion to nitrate via a peroxynitrite intermediate. Here we use RR spectroscopy of rapid freeze quench (RFQ) samples to demonstrate that the millisecond intermediate previously thought to be a Fe(III)-peroxynitrite is in fact a Fe(III)-nitrato complex. We show that DevS also efficiently catalyzes this reaction which would provide MTB with another route for resistance to NO.

Identifier

doi:10.6083/M4SN06X3

Division

Div. of Environmental & Biomolecular Systems

School

School of Medicine

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.