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Title:Studies on the cytochrome bd-type oxygen reductase superfamily and the discovery of a novel nitric oxide reductase
Author(s):Murali, Ranjani
Director of Research:Gennis, Robert B.
Doctoral Committee Chair(s):Gennis, Robert B.
Doctoral Committee Member(s):Nair, Satish K.; Tajkhorshid, Emad; Crofts, Antony R.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):cytochrome bd
heme-copper oxidase
caldivirga maquilingensis
nitric oxide reductase
Abstract:Respiration is the process by which an organism utilizes the reducing equivalents (electrons) produced during catabolism to create an electrochemical potential across the cellular membrane, which can then be expended to produce ATP. Living organisms, bacteria and archaea in particular have discovered numerous ways to respire using highly conserved enzyme superfamilies whose members can couple the free energy released during redox reactions to the creation of proton motive force. Combined with biochemical studies, the evolution of these superfamilies deduced from bioinformatics is very informative about the structural and functional features that are most useful to conserve energy by the creation of proton motive force. This work investigates two important enzyme superfamilies whose members are responsible for virtually all aerobic respiration on the planet– the bd-type oxygen reductase superfamily and the heme-copper oxidoreductase superfamily. The bd-type oxygen reductases are present in bacteria and archaea and catalyze the 4-electron reduction of oxygen to water. Electrons from membrane bound quinols first reduce a low spin heme b from which they are transferred to a di-heme active site where where oxygen is reduced to water. Energy is conserved by the transfer of protons from the cytoplasm to the active site which is located near the periplasmic surface. E. coli has two bd-type quinol oxidases, called cytochrome bd-I and cytochrome bd-II. It had previously been shown that cytochrome bd-I generates a proton motive force, but work from another group claimed that this was not the case for bd-II. As part of this work, it was demonstrated that bd-II functions identically to bd-I and generates a membrane voltage. In a second project dealing with the cytochrome bd superfamily, genomic sequence analysis of cytochrome bd revealed a subfamily of distinct enzymes present in archaea. One of these archael cytochrome bds, was heterologously expressed in Escherichia coli, purified and characterized. This enzyme was shown to have three hemes b instead of two hemes b and one heme d, as is typical of enzymes of this superfamily. The enzyme is highly active, using ubiquinol as substrate. This is the first example in which a member of the cytochrome bd superfamily lacking heme d has been isolated and characterized. A third project involving the importance of a glutamic acid residue in the active site of Escherichia coli cytochrome bd has also been discussed. The heme-copper oxidoreductase (HCO) superfamily contains many proton pumping oxygen reductases as well as several subfamilies of nitric oxide reductases (NORs) which convert nitric oxide to nitrous oxide. These NORs perform different chemistry than the HCOs but have a very similar protein structure and a similar active site. Significantly, the active site of purified NORs contain a Fe atom instead of the copper in the oxygen redutases. Using bioinformatics methods, a distinct new clade of NORs was identified that was previously not recognized. These enzymes have been assumed reduce nitric oxide and were thus named eNORs. One member of this clade is encoded in the genome of Rhodothermus marinus, a thermophilic bacteroidete. Rhodothermus marinus was shown to possess a nitric oxide reductase by measuring the accumulation of nitrous oxide in the containers in which the bacteria is grown on nitrate. Then the enzyme was purified and characterized and confirmed to be a nitric oxide reductase. This confirms for the first time a large group of enzymes, previously not identified, that participate in denitrification in many prokaryotes.
Issue Date:2016-04-18
Rights Information:Copyright 2016 Ranjani Murali
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05

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