Ändra sökning
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Insights into nitric oxide reductase function from a FeB depleted variant
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biokemi och molekylärbiologi Biofysik
Forskningsämne
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-167422OAI: oai:DiVA.org:su-167422DiVA, id: diva2:1300270
Tillgänglig från: 2019-03-28 Skapad: 2019-03-28 Senast uppdaterad: 2019-03-29Bibliografiskt granskad
Ingår i avhandling
1. Assembly and Function of Nitric Oxide Reductase from Paracoccus denitrificans
Öppna denna publikation i ny flik eller fönster >>Assembly and Function of Nitric Oxide Reductase from Paracoccus denitrificans
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study.

Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR.

Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this protein to investigate the role of FeB in the mechanism of nitric oxide and oxygen reduction of cNOR and compared our results to computational studies of the enzyme published recently.

In vitro studies of membrane proteins, such as cNOR, are challenging because their function often depends on the interaction with a biological membrane and specific phospholipids. We used two different membrane mimetic systems, lipid nanodiscs and proteoliposomes, to study the effect of a membrane environment on the function of detergent-solubilized cNOR. Our results indicate that the membrane bilayer of lipid nanodiscs and proteoliposomes, even when assembled using the same lipids, has different properties with measurable effects on cNOR function.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. s. 70
Nyckelord
non-heme iron, cofactor, heme-copper oxidase, AAA+, cNOR, chaperone, metal ion insertion, MoxR, nor accessory genes, FeB, liposomes, membrane mimetics, nanodiscs, reconstitution, enzymatic mechanism, NO reduction, O2 reduction
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
biokemi
Identifikatorer
urn:nbn:se:su:diva-167439 (URN)978-91-7797-636-3 (ISBN)978-91-7797-637-0 (ISBN)
Disputation
2019-05-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Tillgänglig från: 2019-04-23 Skapad: 2019-03-28 Senast uppdaterad: 2019-04-09Bibliografiskt granskad

Open Access i DiVA

Fulltext saknas i DiVA

Sök vidare i DiVA

Av författaren/redaktören
Kahle, MaximilianBlomberg, Margareta R. A.Ädelroth, Pia
Av organisationen
Institutionen för biokemi och biofysikInstitutionen för organisk kemi
Biokemi och molekylärbiologiBiofysik

Sök vidare utanför DiVA

GoogleGoogle Scholar

urn-nbn

Altmetricpoäng

urn-nbn
Totalt: 6 träffar
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf