Architecture of NarGH Reveals a Structural Classification of Mo-bisMGD Enzymes

Abstract

The structure of the catalytic and electron-transfer subunits (NarGH) of the integral membrane protein, respiratory nitrate reductase (Nar) has been determined to 2.0 A resolution revealing the molecular architecture of this Mo-bisMGD (molybdopterin-guanine-dinucleotide) containing enzyme which includes a previously undetected FeS cluster. Nar, together with the related enzyme formate dehydrogenase (Fdh-N), is a key enzyme in the generation of proton motive force across the membrane in Escherichia coli nitrate respiration. A comparative study revealed that Nar and Fdh-N employ different approaches for acquiring substrate, reflecting different catalytic mechanisms. Nar uses a very narrow and nonpolar substrate-conducting cavity with a nonspecific substrate binding site, whereas Fdh-N accommodates a wider, positively charged substrate-conducting cavity with a more specific substrate binding site. The Nar structure also demonstrates the first example of an Asp side chain acting as a Mo ligand providing a structural basis for the classification of Mo-bisMGD enzymes.

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…The coordination of metal sites by side-chain carboxylates from aspartic and glutamic acid molecules has been the subject of several studies ( [36] and references therein). These studies showed that carboxylate groups present a high flexibility of coordination, as suggested by Jormakka et al [26].…”

“…In 2003 the whole complex NarGHI was reported, at a resolution of 1.9 Å [25], and in 2004 the heterodimer NarGH was solved to 2.0 Å resolution [26]. The heterotrimer Nar-GHI comprises a catalytic subunit (NarG, 140 kDa), a subunit containing four [4Fe-4S] centers (NarH, 58 kDa), and a membrane-bound heme b subunit (NarI, 20 kDa) (Fig.…”

“…This is the first time that an Asp-Mo coordination is observed in bis-MGD enzymes. In addition, while the carboxylate group acts as a bidentate ligand in the complete NarGHI structure (distances of $1.9 Å and $2.4 Å for Mo-OD1 and Mo-OD2, respectively) [25], in the NarGH structure Asp222 makes only one bond to the Mo atom [26]. The sixth coordination position is occupied by an oxo group (Mo=O%1.8 Å ) which is absent in the NarGHI structure.…”

“…EPR studies performed in E. coli clearly showed the typical resonances for the 3Fe-4S cluster and two EPR signals, which were attributed to two different conformations of a single Fe/S cluster, but the EPR properties of the remaining Fe/S centers could not be determined precisely. Jormakka et al [26] hypothesized that the Fe/S cluster in the NarG subunit should be both a high-redox potential center, because of the polar environment around it, and a high-spin center, to explain the nondetection of this center by EPR. However, as seen below, [4Fe-4S] centers with a similar environment but with low redox potentials were found in all the proteins described in this review.…”

“…The reduction of nitrate to nitrite by the Nap periplasmic pathway is considered to be a dissimilatory one, since it is not coupled to the generation of a proton gradient across the membrane. In contrast, the respiratory nitrate reduction pathway involves a membranebound protein complex composed of three subunits, NarGHI from E. coli [25,26], which is involved in the redox loop of the proton motive force across biomembranes. NarG is the catalytic subunit and the whole complex is oriented to the cytoplasm.…”