Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

Abstract

In the absence of oxygen and in the presence of nitrate, Escherichia coli induces the production of two energy-converting enzymes: formate dehydrogenase-N (FdnGHI) and dissimilatory nitrate reductase A (NarGHI).3 These two complexes cooperate in generating a proton motive force through the redox loop mechanism as originally envisaged by Peter Mitchell in his chemiosmotic hypothesis (1). The separation of positive and negative charges across the cytoplasmic membrane is achieved through electron transfer from the formate oxidation site in the periplasm to the nitrate reduction site located on the cytoplasmic space with mena-or ubiquinone/quinol cycling between them (2, 3). The heterotrimeric NarGHI complex is composed of (i) a nitrate-reducing subunit NarG containing a Mo-bis-MGD cofactor (Moco) and a [4Fe-4S] cluster FeS0 with unusual His coordination (4, 5), (ii) an electron transfer subunit NarH carrying four FeS clusters (6), and (iii) a membrane anchor subunit NarI containing two b-type hemes termed b D and b P to indicate their distal and proximal positions with respect to the nitratereducing site (7-9). These prosthetic groups define an electron transfer pathway from a periplasmically oriented quinol oxidation site (Q D ) to a cytoplasmically oriented nitrate reduction site.Although considerable research has been devoted to nitrate reductase functioning, only partial information has been gained about the number, the structure, and the specificity of quinol binding sites within NarGHI. For instance, some studies have reported on mena-and ubiquinol analogue binding (10 -20), whereas the molecular details of the interaction between natural quinols and NarGHI remain to be established.Despite the absence of bound quinones in the available high resolution structures of NarGHI (7,20), the crystal structure of the enzyme in complex with pentachlorophenol (PCP), an inhibitor of the quinol oxidase activity, has been determined (20). Based on mutagenesis data, biochemical analyses, and molecular modeling, a model of the Q D quinol binding site has been proposed (20). In this working model, a quinone carbonyl group interacts with the protein via a hydrogen bond to a histidine residue (His-66), which is one of the axial ligands of heme b D . Molecular modeling of a menaquinone in the PCP binding site suggested that the opposite carbonyl group could form a hydrogen bond to . Additionally, an elongated

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

“…Indeed, no evidence for the transfer of a measurable spin density on any other nuclei than that mentioned above was found. Thus, we tentatively proposed that a water-mediated interaction is formed between MSQ D (or USQ D ) and Lys-86, consistent with the latter being involved in reactivity toward quinols (13). Moreover, we have recently shown that a cardiolipin molecule specifically bound to the complex is necessary for quinol substrate fixation at the Q D site, probably through the action of one of its acyl chains located in the vicinity of His-66 (15).…”

“…The functionality of NarGHI in our samples was confirmed spectrophotometrically by measuring the quinol:nitrate oxidoreductase activity. Stabilization of the semiquinone at the Q D site was achieved through redox titrations under the same conditions as those used in our previous works (10,11,13). Redox potentials are given in the text with respect to the standard hydrogen electrode.…”

“…1A). The weakness of this effect is due to the primary contribution to the line shape of the g-tensor anisotropy, which was previously resolved using numerical simulation of the MSQ D Q-band EPR spectrum (10 (12,13). In addition to these 14 N signals, several cross-features from protons symmetrically positioned with respect to the 1 H Zeeman frequency ( I ( 1 H) ϳ14.7 MHz) are clearly resolved in the 10 -20-MHz frequency range in the (ϩ,ϩ) quadrant of these spectra ( Fig.…”

“…Spectra were measured at 90 K to avoid contamination from fast relaxing metal centers such as FeS centers in NarGHI (13). For the two-pulse experiments (/2--), the echo intensity was measured as a function of magnetic field at fixed time interval between the two microwave pulses for field sweep ESE or as a function of at a fixed magnetic field value for two-pulse ESEEM.…”

Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

“…Care however needs to be taken (see ref. 80 for an example where this was not considered) as this is clearly an underestimation due to the rest (0.8) of the partial spin density spread over the rest of the quinone head group moiety and indeed by taking all of this spin density distribution into consideration this results in an O-N distance of 2.5-2.6 A which indicates an H-bond length of 1.5-1.6 A.…”