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

Thomas F. Prisner,Burkhard Endeward,Axel Magalon,Rodrigo Arias-Cartin,Stéphane Grimaldi,Pascal Lanciano,Sevdalina Lyubenova,Bruno Guigliarelli

doi:10.1074/jbc.m109.060251

Abstract

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Highlights

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 bD and bP to indicate their distal and proximal positions with respect to the nitratereducing site [7,8,9]
On the basis of these observations, we concluded that the menasemiquinone radical is located in the vicinity of heme bD and that Lys-86 is required for its stabilization [19]
X-band Pulsed EPR of Native and 15N-Labeled NarGHI— Our previous studies revealed that an EPR-detectable menasemiquinone intermediate can be generated in the wild-type nitrate reductase A by direct chemical reduction of purified E. coli NarGHI-enriched membrane fractions [18], the maximal SQ concentration being reached at a redox potential around Ϫ100 mV at pH 7.5 [18]

Summary

Introduction

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 bD and bP to indicate their distal and proximal positions with respect to the nitratereducing site [7,8,9]. Biochemical analyses, and molecular modeling, a model of the QD 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 bD. The MSQ stability constant measured in NarGHI (Ks ϳ70) is the largest measured so far in respiratory complexes stabilizing semiquinone species [21,22,23] This semiquinone species is present in a mutant lacking the proximal heme bP, it cannot be detected in variants lacking the distal heme or upon substitution of the surrounding Lys-86 residue by an Ala (NarGHIK86A) [19]. The molecular factors responsible for the high MSQD stabilization and the role of this stability during the enzyme mechanism remain to be elucidated

Results

Discussion

Conclusion