Date

March 2011

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

The ultimate goal of this research is to define the mechanisms by which metalloproteins reduce NO to N[subscript 2]O. This reaction is an obligate step in bacterial denitrification, and it also provides microorganisms resistance to nitrosative stresses. We have characterized two prominent families of enzymes that catalyze this reaction. Terminal oxidases are members of heme/copper oxidase superfamily and are evolutionally related to denitrifying heme/non-heme NO reductase enzymes (NORs). Despite differences in recognized function and active site metal composition, these enzymes exhibit cross reactivity with NORs showing oxidase activity and several terminal oxidases capable of reducing NO to N[subscript 2]O. Here, we use low temperature photolysis combined with UV-vis, FTIR, resonance Raman (RR), and EPR spectroscopies to determine how NO interacts with the metal centers of cytochrome ba[subscript 3] from Thermus thermophilus, bo[subscript 3] from Escherichia coli, and bioengineered models of NORs in myoglobin scaffold. Our results show that in all cases, the first NO molecule binds to the heme iron(II) and that the binding of a second NO to the distal metal center is not an essential step of the NO reductase activity. Indeed, while a side-on copper-nitrosyl complex can be trapped in ba[subscript 3], none of the enzymes studied show the formation of an end-on distal metal-NO complex. Thus, the role of the distal metal center is proposed to be limited to the stabilization of a heme-hyponitrite species via electrostatic interaction. This NO reduction mechanism is described as a cis-heme reaction route and is supported by theoretical calculations. We also investigated the NO reductase activity of flavodiiron proteins (FDPs) using UV-vis, FTIR, RR, and EPR spectroscopies. The close vicinity of the flavin mononucleotide cofactor (FMN) to the diiron site has led to the proposal of a superreduction mechanism where [Fe[superscript II]-NO][subscript 2] complex is reduced by FMN. To distinguish the role that FMN and diiron center play in catalysis, we prepared an FMN-free FDP (deflavo-FDP). Our experiments show that the reduced diiron center in deflavo-FDP is capable of a single reducing turnover of NO to N2O. Furthermore, stoichiometric addition of NO to the reduced deflavo-FDP as well as holo-FDP results in the formation of a diiron-mononitrosyl complex Fe[superscript II] • Fe[superscript II]-NO as the first intermediate in the catalysis. These results support a catalytic route for NO reductase in FDPs where the diiron site reduces NO to N[subscript 2]O and the role of the FMN cofactor is to reduce the oxidized diiron site.

Identifier

doi:10.6083/M4G44N8H

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.