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The insertion of the non-heme Fe-B cofactor into nitric oxide reductase from P. denitrificans depends on NorQ and NorD accessory proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-4165-9277
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Number of Authors: 42018 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, no 10, p. 1051-1058Article in journal (Refereed) Published
Abstract [en]

Bacterial NO reductases (NOR) catalyze the reduction of NO into N2O, either as a step in denitrification or as a detoxification mechanism. cNOR from Paracoccus (P.) denitrificans is expressed from the norCBQDEF operon, but only the NorB and NorC proteins are found in the purified NOR complex. Here, we established a new purification method for the P. denitrificans cNOR via a His-tag using heterologous expression in E. coli. The His-tagged enzyme is both structurally and functionally very similar to non-tagged cNOR. We were also able to express and purify cNOR from the structural genes norCB only, in absence of the accessory genes norQDEF. The produced protein is a stable NorCB complex containing all hemes and it can bind gaseous ligands (CO) to heme b(3), but it is catalytically inactive. We show that this deficient cNOR lacks the nonheme iron cofactor Fe B . Mutational analysis of the nor gene cluster revealed that it is the norQ and norD genes that are essential to form functional cNOR. NorQ belongs to the family of MoxR P-loop AAA + ATPases, which are in general considered to facilitate enzyme activation processes often involving metal insertion. Our data indicates that NorQ and NorD work together in order to facilitate non-heme Fe insertion. This is noteworthy since in many cases Fe cofactor binding occurs spontaneously. We further suggest a model for NorQ/D-facilitated metal insertion into cNOR.

Place, publisher, year, edition, pages
2018. Vol. 1859, no 10, p. 1051-1058
Keywords [en]
Iron, AAA ATPases, cNOR, Metal ion insertion, nor accessory genes, His-tag, E. coli, Chaperone, Protein assembly, MoxR
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-161172DOI: 10.1016/j.bbabio.2018.05.020ISI: 000444929300007PubMedID: 29874552OAI: oai:DiVA.org:su-161172DiVA, id: diva2:1261727
Available from: 2018-11-08 Created: 2018-11-08 Last updated: 2019-03-29Bibliographically approved
In thesis
1. Assembly and Function of Nitric Oxide Reductase from Paracoccus denitrificans
Open this publication in new window or tab >>Assembly and Function of Nitric Oxide Reductase from Paracoccus denitrificans
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 70
Keywords
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
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-167439 (URN)978-91-7797-636-3 (ISBN)978-91-7797-637-0 (ISBN)
Public defence
2019-05-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

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

Available from: 2019-04-23 Created: 2019-03-28 Last updated: 2020-05-15Bibliographically approved

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