Tagged: NAR

Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR, 14,15N HYSCORE, and DFT Study

Julia Rendon, Frédéric Biaso, Pierre Ceccaldi, René Toci, Farida Seduk, Axel Magalon, Bruno Guigliarelli, and Stéphane Grimaldi. Inorg. Chem., 2017, 56 (8), pp 4422–4434. DOI: 10.1021/acs.inorgchem.6b03129

Combining multiple isotope-enrichment strategies in 98Mo and 15N nuclei together with EPR, HYSCORE spectroscopy, and DFT modeling, we propose a structural model of the low-pH Mo(V) species in respiratory nitrate reductase that implies coordination of the metal by a monodentate Asp222 ligand and a hydroxyl moiety. Furthermore, we unveil the peculiar involvement of the conserved Asn52 to the H-bond network around the Mo-cofactor in both low- and high-pH species.

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Redox cofactor insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

Rodrigo Arias-Cartin, Pierre Ceccaldi, Barbara Schoepp-Cothenet, Klaudia Frick, Jean-Michel Blanc, Bruno Guigliarelli, Anne Walburger, Stéphane Grimaldi, Thorsten Friedrich, Véronique Receveur-Brechot, Axel Magalon. Scientific Reports 6, Article number: 37743 (2016) http://dx.doi.org/10.1038/srep37743

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The prokaryotic Mo/W-bisPGD enzymes family: A catalytic workhorse in bioenergetic.

Grimaldi S, Schoepp-Cothenet B, Ceccaldi P, Guigliarelli B, Magalon A.

Over the past two decades, prominent importance of molybdenum-containing enzymes in prokaryotes has been put forward by studies originating from different fields. Proteomic or bioinformatic studies underpinned that the list of molybdenum-containing enzymes is far from being complete with to date, more than fifty different enzymes involved in the biogeochemical nitrogen, carbon and sulfur cycles. In particular, the vast majority of prokaryotic molybdenum-containing enzymes belong to the so-called dimethylsulfoxide reductase family. Despite its extraordinary diversity, this family is characterized by the presence of a Mo/W-bis(pyranopterin guanosine dinucleotide) cofactor at the active site. This review highlights what has been learned about the properties of the catalytic site, the modular variation of the structural organization of these enzymes, and their interplay with the isoprenoid quinones. In the last part, this review provides an integrated view of how these enzymes contribute to the bioenergetics of prokaryotes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

Cardiolipin binding in bacterial respiratory complexes: Structural and functional implications.

Arias-Cartin R, Grimaldi S, Arnoux P, Guigliarelli B, Magalon A.

Biochim Biophys Acta. 2012 Oct;1817(10):1937-49. doi: 10.1016/j.bbabio.2012.04.005. Epub 2012 Apr 17.

The structural and functional integrity of biological membranes is vital to life. The interplay of lipids and membrane proteins is crucial for numerous fundamental processes ranging from respiration, photosynthesis, signal transduction, solute transport to motility. Evidence is accumulating that specific lipids play important roles in membrane proteins, but how specific lipids interact with and enable membrane proteins to achieve their full functionality remains unclear. X-ray structures of membrane proteins have revealed tight and specific binding of lipids. For instance, cardiolipin, an anionic phospholipid, has been found to be associated to a number of eukaryotic and prokaryotic respiratory complexes. Moreover, polar and septal accumulation of cardiolipin in a number of prokaryotes may ensure proper spatial segregation and/or activity of proteins. In this review, we describe current knowledge of the functions associated with cardiolipin binding to respiratory complexes in prokaryotes as a frame to discuss how specific lipid binding may tune their reactivity towards quinone and participate to supercomplex formation of both aerobic and anaerobic respiratory chains. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR.

Grimaldi S, Arias-Cartin R, Lanciano P, Lyubenova S, Szenes R, Endeward B, Prisner TF, Guigliarelli B, Magalon A.

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (QD) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the QD site (MSQD) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with 1H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQD binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme bD. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the QD site are discussed, in light of the unusually high thermodynamic stability of MSQD.

Cardiolipin-based respiratory complex activation in bacteria.

Arias-Cartin R, Grimaldi S, Pommier J, Lanciano P, Schaefer C, Arnoux P, Giordano G, Guigliarelli B, Magalon A.

Proc Natl Acad Sci U S A. 2011 May 10;108(19):7781-6. doi: 10.1073/pnas.1010427108. Epub 2011 Apr 25.

Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid–protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme bD center and is responsible for structural adjustments of bD and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.

HYSCORE Evidence That Endogenous Mena- and Ubisemiquinone Bind at the Same Q Site (QD) of Escherichia coli Nitrate Reductase A

Arias-Cartin R, Lyubenova S, Ceccalci P, Prisner T, Magalon A, Giugliarelli B, Grimaldi S

J Am Chem Soc. 2010 May 5;132(17):5942-3. doi: 10.1021/ja1009234.

Through the use of an Escherichia coli strain deficient in menaquinone biosynthesis, purified nitrate reductase A (NarGHI)-enriched inner membrane vesicles were titrated and monitored by electron paramagnetic resonance (EPR) spectroscopy, revealing the formation of protein-bound ubisemiquinone (USQ) species. Two-dimensional ESEEM (HYSCORE) experiments on these radicals revealed the same magnetic interaction with an 14N nucleus as found for menasemiquinone stabilized at the QD site of E. coli NarGHI and assigned to His66 Nδ, a distal heme axial ligand. Moreover, this signature was lost in the NarGHIH66Y mutant, which is known to be unable to react with quinols. These findings demonstrate that NarGHI-bound USQ can be formed and detected by EPR. They also provide the first direct experimental evidence for similar binding of natural menasemiquinones and ubisemiquinones within the same protein Q site of NarGHI.

Direct evidence for nitrogen ligation to the high-stability semiquinone intermediate in Escherichia coli nitrate reductase A

Grimaldi S, Arias-Cartin R, Lanciano P, Lyubenova S, Endeward B, Prisner TF, Magalon A, Guigliarelli B

J Biol Chem. 2010 Jan 1;285(1):179-87. doi: 10.1074/jbc.M109.060251. Epub 2009 Nov 5.

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 (QD) located in the vicinity of the distal heme bD. Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. 14N and 15N ESEEM and HYSCORE measurements carried out at X-band (∼9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with 15N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant Aiso(14N) ∼0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using 14N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, ∼3.4 GHz), the 14N nuclear quadrupolar parameters of the interacting nitrogen nucleus (κ = 0.49, η = 0.50) were determined and correspond to those of a histidine Nδ, assigned to the heme bD ligand His-66 residue. Moreover S-band 15N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T(15N) = 0.16 MHz). A distance of ∼2.2 Åbetween 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 QD site of NarGHI.

New method for the spin quantitation of [4Fe-4S](+) clusters with S = (3)/(2). Application to the FS0 center of the NarGHI nitrate reductase from Escherichia coli.

Lanciano P, Savoyant A, Grimaldi S, Magalon A, Guigliarelli B, Bertrand P

In conventional analyses of g ≈ 5 signals given by [4Fe−4S]+ clusters with S = 3/2, the effective g values that cannot be measured in the electron paramagnetic resonance (EPR) spectrum are deduced from rhombograms calculated by assuming that the matrix is isotropic with gx = gy = gz = 2.00. We have shown that when the two low-field peaks corresponding to the Kramers doublets are visible in the spectrum, a new, independent piece of information about the system can be obtained by studying the temperature dependence of the ratio of the area under these peaks. By applying this method to the g ≈ 5 signals displayed by NarGHI nitrate reductase, we were able to determine all the parameters of the spin Hamiltonian of FS0 centers with S = 3/2 and to measure accurately their number. Our results indicate that simple analyses based on the assumption of an isotropic matrix can give rise to very large errors.