NosL is a dedicated copper chaperone for assembly of the CuZ center of nitrous oxide reductase. Issue 19 (18th April 2019)

• Record Type: Journal Article
• Authors: Bennett, Sophie P.; Soriano-Laguna, Manuel J.; Bradley, Justin M.; Svistunenko, Dimitri A.; Richardson, David J.; Gates, Andrew J.; Le Brun, Nick E.
• Is Part Of: Chemical science. Volume 10:Issue 19(2019)
• Page Start: 4985
• Page End: 4993
• Publication Date: 2019-04-18
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c9sc01053j
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : The Cu(i )-binding protein NosL functions specifically as an assembly factor for the unique CuZ centre of nitrous oxide reductase (N2 OR). Abstract : Nitrous oxide reductase (N2 OR) is the terminal enzyme of the denitrification pathway of soil bacteria that reduces the greenhouse gas nitrous oxide (N2 O) to dinitrogen. In addition to a binuclear CuA site that functions in electron transfer, the active site of N2 OR features a unique tetranuclear copper cluster bridged by inorganic sulfide, termed CuZ . In copper-limited environments, N2 OR fails to function, resulting in truncation of denitrification and rising levels of N2 O released by cells to the atmosphere, presenting a major environmental challenge. Here we report studies of nosL from Paracoccus denitrificans, which is part of the nos gene cluster, and encodes a putative copper binding protein. A Paracoccus denitrificans Δ nosL mutant strain had no denitrification phenotype under copper-sufficient conditions but failed to reduce N2 O under copper-limited conditions. N2 OR isolated from Δ nosL cells was found to be deficient in copper and to exhibit attenuated activity. UV-visible absorbance spectroscopy revealed that bands due to the CuA center were unaffected, while those corresponding to the CuZ center were significantly reduced in intensity. In vitro studies of a soluble form of NosL without its predicted membrane anchor showed that it binds one Cu(i ) ion per protein with attomolar affinity, but does not bind Cu(ii ). Together, the data demonstrate that NosL is a copper-binding protein specifically required for assembly of the CuZ center of N2 OR, and thus represents the first characterised assembly factor for the CuZ active site of this key environmental enzyme, which is globally responsible for the destruction of a potent greenhouse gas.