Title page for ETD etd-11597-135857


Type of Document Dissertation
Author Thorsteinsson, Marc Victor III
Author's Email Address mthorste@vt.edu
URN etd-11597-135857
Title Structural and Functional Characterization of Cyanoglobin: A Peripheral Membrane Hemoglobin in Nostoc commune UTEX 584 (Cyanobacteria)
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Bevan, David R.
Ebel, Richard E.
Helm, Richard Frederick
Hess, John L.
Potts, Malcolm Committee Chair
Keywords
  • oxygen
  • hemoglobin
  • nitrogen fixation
Date of Defense 1997-12-03
Availability unrestricted
Abstract
Investigations of the nitrogen fixing (nif) genes in the cyanobacterium Nostoc commune UTEX 584 revealed a gene encoding a hemoprotein, named cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. Cyanoglobin possesses a high oxygen affinity. The study presented here investigated the functional role of cyanoglobin, and encompassed the determination of the kinetic basis for the high oxygen affinity of cyanoglobin through kinetic studies utilizing stopped-flow spectrophotometry and flash photolysis. In addition, studies of cyanoglobin, in the presence of a variety of ligands, employed as structural probes of the distal pocket architecture, are presented. These data are interpreted in terms of structural models of cyanoglobin produced by homology modelling and hemoglobins with known crystal structures. Cyanoglobin coordinated oxygen and a variety of ligands with high rates of association, which explained the high oxygen affinity of cyanoglobin. Cyanoglobin possessed high rates of autoxidation and hemin loss. The ligand binding behavior of cyanoglobin was more similar to leghemoglobin than to sperm whale myoglobin. The ligand binding behavior of cyanoglobin is explained in terms of a highly reactive, and solvent exposed, heme-iron. The 5' region of glbN interacted with NtcA, the global regulator of nitrogen metabolism in cyanobacteria, which may provide an indication of the nitrogen deprivation signal required for cyanoglobin expression in vivo. Finally, the isolation and N-terminal sequencing of a potential cyanoglobin homolog in Anabaena sp. strain PCC 7120 is presented. Collectively, the data obtained in this study may support the model of cyanoglobin function described by Hill, et al., that cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.
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