A [3Cu:2S] cluster provides insight into the assembly and function of the CuZ site of nitrous oxide reductase. Issue 9 (19th January 2021)

• Record Type: Journal Article
• Authors: Zhang, Lin; Bill, Eckhard; Kroneck, Peter M. H.; Einsle, Oliver
• Is Part Of: Chemical science. Volume 12:Issue 9(2021)
• Page Start: 3239
• Page End: 3244
• Publication Date: 2021-01-19
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d0sc05204c
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : Variants of all seven histidine ligands of the [4Cu:2S] active site of nitrous oxide reductase mostly result in loss of the metal site. However, a H382A variant retains a [3Cu:2S] cluster that hints towards a structural flexibility also present in the intact site. Abstract : Nitrous oxide reductase (N2 OR) is the only known enzyme reducing environmentally critical nitrous oxide (N2 O) to dinitrogen (N2 ) as the final step of bacterial denitrification. The assembly process of its unique catalytic [4Cu:2S] cluster CuZ remains scarcely understood. Here we report on a mutagenesis study of all seven histidine ligands coordinating this copper center, followed by spectroscopic and structural characterization and based on an established, functional expression system for Pseudomonas stutzeri N2 OR in Escherichia coli . While no copper ion was found in the CuZ binding site of variants H129A, H130A, H178A, H326A, H433A and H494A, the H382A variant carried a catalytically inactive [3Cu:2S] center, in which one sulfur ligand, SZ2, had relocated to form a weak hydrogen bond to the sidechain of the nearby lysine residue K454. This link provides sufficient stability to avoid the loss of the sulfide anion. The UV-vis spectra of this cluster are strikingly similar to those of the active enzyme, implying that the flexibility of SZ2 may have been observed before, but not recognized. The sulfide shift changes the metal coordination in CuZ and is thus of high mechanistic interest.