Abstract
Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br 2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br 2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.
Highlights
Understanding catalysis by enzymes underpinned by high-resolution three-dimensional structures has attracted major attention from structural enzymologists who have catalyzed the advances in experimental capabilities by continuing to push the capabilities of synchrotron radiation (SR) crystallography improving brilliance of x-ray beams and detectors and have recently opened the possibility of obtaining damage-free, free from radiation-induced chemistry (FRIC), structures
We have generated the product in situ by using substrate- soaked crystals with full occupancy of substrate at the catalytic type 2 Cu (T2Cu) site and treating them with a chemical reductant to initiate the turnover. This has allowed characterization of the stable nitrosyl intermediate with “side-on” binding unequivocally refuting the hypothesis that “the proton-coupled electron transfer to the nitrite-bound T2Cu leads to reduction of nitrite to form NO, which is released without forming a Cu-nitrosyl species” [22,23,24]
The observation of an in situ produced copper nitrosyl species in both the SRX and x-ray free-electron laser (XFEL) FRIC structures at ~1.2 Å unambiguously demonstrates this as a catalytically relevant reaction intermediate, refuting the hypothesis that “the proton-coupled electron transfer (PCET) to the nitrite-b ound T2Cu leads to reduction of nitrite to form NO, which is released without forming a Cu-nitrosyl species” [22,23,24]
Summary
Understanding catalysis by enzymes underpinned by high-resolution three-dimensional structures has attracted major attention from structural enzymologists who have catalyzed the advances in experimental capabilities by continuing to push the capabilities of synchrotron radiation (SR) crystallography improving brilliance of x-ray beams and detectors and have recently opened the possibility of obtaining damage-free, free from radiation-induced chemistry (FRIC), structures We have applied these advanced methods to obtain the most accurate [SR structures at resolutions of ~1 Å enabling unrestrained, SHELX refinement for any copper-containing nitrite reductase (CuNiR)] and damage-free x-ray free-electron laser (XFEL)–FRIC structures, in the as-isolated, substrate-bound, and product-bound states, again to high resolutions of 1.3 Å. In addition to nitrogen fixation, Rhizobia are able to use fixed nitrogen, in the form of nitrate, as a replacement for dioxygen during respiratory adenosine 5′-triphosphate syn-
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