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Title:Characterization of the quinol nitric oxide reductase from Peresephonella marina
Author(s):Sheraden, Paige
Advisor(s):Gennis, Robert B.
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:M.S.
Genre:Thesis
Subject(s):nitric oxide reductase
quinol nitric oxide reductase (qNOR)
Abstract:qNOR is a nitric oxide reductase in the heme-copper oxidoreductase superfamily that catalyzes the two electron reduction of NO to N2O using electrons derived from quinol. In this study, we heterologously expressed and characterized the qNOR from the thermophilic, denitrifying bacteria Persephonella marina. The reaction rate of this qNOR varied linearly from 0.22±0.05 at 42°C to 2.3±0.3 at 60°C. We also report the first Km for a purified qNOR, and first activation energy of any NOR, 3.1±0.6 μM NO and 114 kJ/mol respectively. qNORs have three highly conserved glutamate residues in their active sites. Mutagenesis studies demonstrate that E476 is likely the FeB ligand, E480 helps maintain the electronegative environment of the active site, and that E545 is involved in the proton entry pathway. A binding site for the inhibitor HQNO was identified in the recently reported qNOR structure (Matsumoto et al 2012). Using sequence analysis and mutagenesis we were able to confirm that the quinol binding site is comprised of residues H295, R705, and D709, which are the same residues that bind HQNO. Also in this same crystal structure, a hydrophilic channel connecting the cytoplasm to the active site was resolved and hypothesized to be a proton entry channel, similar to the K-channel of heme-copper oxidases. However, the channel was not confirmed for active protein. In this study, we also investigated proton entry pathways in qNOR. We found that the residues lining the proposed cytoplasmic proton entry channel were not conserved and mutations of the polar residues to leucine had little effect on activity. These findings indicate that this channel is not important for active enzyme. We also attempted to elucidate a complete periplasmic proton entry pathway, but as most mutations were made near the active site heme b3, a total lack of activity in most mutants was difficult to interpret. However, the near complete conservation of several polar residues between the heme b3 iii propionate and the active site, allows us to hypothesize their importance in delivering protons to the active site once they reach the level of the heme propionate.
Issue Date:2013-05-24
URI:http://hdl.handle.net/2142/44093
Rights Information:Copyright 2013 Paige Sheraden
Date Available in IDEALS:2013-05-24
Date Deposited:2013-05


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