Reverse protein engineering of a novel 4‐domain copper nitrite reductase reveals functional regulation by protein–protein interaction

Abstract

Cu‐containing nitrite reductases that convert NO2‐ to NO are critical enzymes in nitrogen‐based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4‐domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2‐domain CuNiR (Br2DNiR). We report the first enzymatic studies of a novel 4‐domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3‐ and 2‐domain variants, and Br2DNiR revealing up to ~ 500‐fold difference in catalytic efficiency in comparison with classical 2‐domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self‐contained donor–acceptor system, we demonstrate that 4‐domain BrNiR utilizes N‐terminal tethering for downregulating enzymatic activity instead. Both Br2DNiR and an engineered 2‐domain variant of BrNiR (Δ(Cytc‐Cup) BrNiR) have 3 to 5% NiR activity compared to the well‐characterized 2‐domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc‐Cup) BrNiR and Br2DNiR with classical 2‐domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4‐domain CuNiR and its engineered 3‐ and 2‐domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. Enzyme Copper‐containing nitrite reductase (CuNiR) (EC 1.7.2.1). Database The atomic coordinate and structure factor of Δ(Cytc‐Cup) BrNiR and Br2DNiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6THE and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6THF, respectively.

Copper nitrite reductase from Sinorhizobium meliloti 2011: Crystal structure and interaction with the physiological versus a nonmetabolically related cupredoxin‐like mediator

“…Hydrophobic residues substituting Asn107 were also observed in the two‐domain rhizobial NirKs from Bradyrhizobium sp. ORS 375 ( Br 2D NirK) and B. japonicum USDA110 ( Bj NirK), both showing turnover numbers lower than those of Af NirK and Ac NirK 17,37 . In line with this observation, Sm NirK has a hydrophobic Val residue at this position and, as reported in our previous work, MV‐dependent activity of Sm NirK shows only 12% of the Ax NirK maximum turnover 22 .…”

An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures

“…We have recently identified a blue CuNiR from Bradyrhizobium ORS 375 of the order Rhizobiales (Br 2D NiR) (21). The catalytic efficiency of Br 2D NiR was found to be substantially lower than two of the well-studied two-domain CuNiRs representing the blue (AxNiR) and green (AcNiR) subclasses, despite a sequence identity of ~70% (21). The blue crystals of Br 2D NiR proved amenable to soaking of substrate and yielded highly diffracting crystals, providing a unique opportunity to obtain atomic-resolution structures of up to 1 Å with synchrotron x-rays and damage-free FRIC structures using XFEL, to ~1.3 Å resolution of as-isolated, nitrite-and nitric oxide-bound states.…”

In Silico Analysis of the Enzymes Involved in Haloarchaeal Denitrification

“…It has been identified in species from Rhizobiales order, such as Bradyrhizobium sp. ORS 375 [62]. They contain the core domain of "classical" two-domains NirK (cupredoxin domain) to which both a cytochrome c and cupredoxin domain are tethered at the N terminus [62].…”

“…ORS 375 [62]. They contain the core domain of "classical" two-domains NirK (cupredoxin domain) to which both a cytochrome c and cupredoxin domain are tethered at the N terminus [62]. Recent studies reported a division of NirK sequences into two different phylogenetic clades.…”

“…These coordination patterns (His 2 -Cys-Met and His 3 -H 2 O) have been detected and conserved in others twodomain NirK, three-domain NirK and four-domain NirK enzymes belonging to Bacteria and Archaea domains such as Nitrosomonas europaea [72], Alcaligenes faecalis [73], Alcaligenes xylosoxidans [74], Ralstonia pickettii [75], Haloarcula marismortui [19] or Bradyrhizobium sp. ORS 375 [62]. The catalytic mechanism of NirK involves Asp and His residues that donate two protons necessary for nitrite to nitric oxide conversion.…”