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Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
Abstract [en]

Bacterial nitric oxide reductases (NORs) catalyse the reduction of two NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the host. Although NORs are members of the heme-copper oxidase superfamily, and thus relatives of proton-pumping O2-reducing enzymes, the best studied NORs, cNORs (cytochrome c dependent), were found to be non-electrogenic.

Here, we focus on another type of NOR, qNOR (quinol-dependent). qNOR from Neisseria meningitidis, a human pathogen, was expressed in Escherichia coli and purified as a stable and highly active NO reductase. Spectroscopic and metal analysis of the purified qNOR showed properties largely similar to those in cNORs. Furthermore, the liposome-reconstituted qNOR showed respiratory control ratios consistently above 2, indicative of an electrogenic reaction. We also exchanged residues in a putative proton pathway leading from the cytoplasm to the active site, but there were no significant effects on either turnover rates or electrogenicity. However, the exchange of a glutamate close to the active site (E-498) yielded drastic effects on turnover. We thus suggest that the N. meningitidis qNOR uses cytoplasmic protons, but that the pathway is rather wide and redundant, narrowing around the glutamate-498.

Emneord [en]
heme-copper oxidase, liposome, proton transfer, respiratory control ratio
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-147210OAI: oai:DiVA.org:su-147210DiVA, id: diva2:1142699
Tilgjengelig fra: 2017-09-20 Laget: 2017-09-20 Sist oppdatert: 2022-02-28bibliografisk kontrollert
Inngår i avhandling
1. Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases
Åpne denne publikasjonen i ny fane eller vindu >>Proton pathways in energy conversion: K-pathway analogs in O2- and NO-reductases
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic.

Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction.

The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. s. 66
Emneord
heme-copper oxidase, cytochrome c oxidase, membrane protein, respiration, electron transfer, proton transfer, redox reaction, metalloprotein, non-heme iron, cytochrome ba3, flow-flash, carbon monoxide, liposome, respiratory control ratio
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-147267 (URN)978-91-7649-986-3 (ISBN)978-91-7649-987-0 (ISBN)
Disputas
2017-11-09, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Tilgjengelig fra: 2017-10-17 Laget: 2017-09-20 Sist oppdatert: 2022-02-28bibliografisk kontrollert

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