The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer

Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer–dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.

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The bacterial respiratory nitric oxide reductase

Biochemical Society Transactions ◽

10.1042/bst0370392 ◽

2009 ◽

Vol 37 (2) ◽

pp. 392-399 ◽

Cited By ~ 43

Author(s):

Nicholas J. Watmough ◽

Sarah J. Field ◽

Ross J. L. Hughes ◽

David J. Richardson

Keyword(s):

Nitric Oxide ◽

Nitrous Oxide ◽

Reaction Mechanism ◽

Proton Transfer ◽

Active Site ◽

Paracoccus Denitrificans ◽

Reductive Coupling ◽

Nitric Oxide Reductase ◽

Cytochrome Bc ◽

Haem Iron

The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.

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Reaction of Carbon Monoxide with the Reduced Active Site of Bacterial Nitric Oxide Reductase†

Biochemistry ◽

10.1021/bi011428t ◽

2001 ◽

Vol 40 (44) ◽

pp. 13361-13369 ◽

Cited By ~ 27

Author(s):

Janneke H. M. Hendriks ◽

Louise Prior ◽

Adam R. Baker ◽

Andrew J. Thomson ◽

Matti Saraste ◽

...

Keyword(s):

Nitric Oxide ◽

Carbon Monoxide ◽

Active Site ◽

Nitric Oxide Reductase

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Inhibition of Vascular Nitric Oxide after Rat Chronic Brain Hypoperfusion: Spatial Memory and Immunocytochemical Changes

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600057 ◽

2005 ◽

Vol 25 (6) ◽

pp. 663-672 ◽

Cited By ~ 78

Author(s):

Jack C. de la Torre ◽

Gjumrakch Aliev

Keyword(s):

Nitric Oxide ◽

Spatial Memory ◽

Memory Function ◽

Critical Role ◽

Artery Occlusion ◽

Carotid Artery Occlusion ◽

Significant Loss ◽

Electron Microscopic ◽

Vessel Occlusion ◽

Common Carotid Artery Occlusion

An aging rat model of chronic brain hypoperfusion (CBH) that mimics human mild cognitive impairment (MCI) was used to examine the role of nitric oxide synthase (NOS) isoforms on spatial memory function. Rats with CBH underwent bilateral common carotid artery occlusion (2-vessel occlusion (2-VO)) for either 26 or 8 weeks and were compared with nonoccluded sham controls (S-VO). The neuronal and endothelial (nNOS/eNOS) constitutive inhibitor nitro-L-arginine methyl ester (L-NAME) 20 mg/kg was administered after 26 weeks for 3 days to 2-VO and S-VO groups and spatial memory was assessed with a modified Morris watermaze test. Only 2-VO rats worsened their spatial memory ability after L-NAME. Electron microscopic immunocytochemical examination using an antibody against eNOS showed 2-VO rats had significant loss or absence of eNOS-containing positive gold particles in hippocampal endothelium and these changes were associated with endothelial cell compression, mitochondrial damage and heavy amyloid deposition in hippocampal capillaries and perivascular region. In the 8-week study, three groups of 2-VO rats were administered an acute dose of 7-NI, aminoguanidine or L-NIO, the relatively selective inhibitors of nNOS, inducible NOS and eNOS. Only rats administered the eNOS inhibitor L-NIO worsened markedly their watermaze performance ( P=0.009) when compared with S-VO nonoccluded controls. We conclude from these findings that vascular nitric oxide derived from eNOS may play a critical role in spatial memory function during CBH possibly by keeping cerebral perfusion optimal through its regulation of microvessel tone and cerebral blood flow and that disruption of this mechanism can result in spatial memory impairment. These findings may identify therapeutic targets for preventing MCI and treating Alzheimer's disease.

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X-ray structure of nitric oxide reductase (cytochrome P450nor) at atomic resolution

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444901017383 ◽

2001 ◽

Vol 58 (1) ◽

pp. 81-89 ◽

Cited By ~ 13

Author(s):

Hideaki Shimizu ◽

Sam-Yong Park ◽

Yoshitsugu Shiro ◽

Shin-ichi Adachi

Keyword(s):

Nitric Oxide ◽

Atomic Resolution ◽

Nitric Oxide Reductase ◽

X Ray

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Dioxygen and nitric oxide reactivity of a reduced heme/non-heme diiron(II) complex [(5L)FeII⋯FeIICl]+. Using a tethered tetraarylporphyrin for the development of an active site reactivity model for bacterial nitric oxide reductase

Inorganica Chimica Acta ◽

10.1016/s0020-1693(99)00404-1 ◽

2000 ◽

Vol 297 (1-2) ◽

pp. 362-372 ◽

Cited By ~ 11

Author(s):

Telvin D. Ju ◽

Amina S. Woods ◽

Robert J. Cotter ◽

Pierre Moënne-Loccoz ◽

Kenneth D. Karlin

Keyword(s):

Nitric Oxide ◽

Active Site ◽

Nitric Oxide Reductase

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Proton transfer in bacterial nitric oxide reductase

Biochemical Society Transactions ◽

10.1042/bst0340188 ◽

2006 ◽

Vol 34 (1) ◽

pp. 188-190 ◽

Cited By ~ 13

Author(s):

U. Flock ◽

J. Reimann ◽

P. Ädelroth

Keyword(s):

Nitric Oxide ◽

Proton Transfer ◽

Structural Model ◽

No Reduction ◽

Paracoccus Denitrificans ◽

Proton Donor ◽

Nitric Oxide Reductase ◽

Reduction Of No ◽

Proton Coupled Electron Transfer ◽

Electron Reduction

The NOR (nitric oxide reductase) from Paracoccus denitrificans catalyses the two-electron reduction of NO to N2O (2NO+2H++2e−→N2O+H2O). The NOR is a divergent member of the superfamily of haem-copper oxidases, oxygen-reducing enzymes which couple the reduction of oxygen with translocation of protons across the membrane. In contrast, reduction of NO catalysed by NOR is non-electrogenic which, since electrons are supplied from the periplasmic side of the membrane, implies that the protons needed for NO reduction are also taken from the periplasm. Thus NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site. The proton pathway has not been identified, and the mechanism and timing of proton transfer during NO reduction is unknown. To address these questions, we have studied the reaction between NOR and the chemically less reactive oxidant O2 [Flock, Watmough and Ädelroth (2005) Biochemistry 44, 10711–10719]. When fully reduced NOR reacts with O2, proton-coupled electron transfer occurs in a reaction that is rate-limited by internal proton transfer from a group with a pKa of 6.6. This group is presumably an amino acid residue close to the active site that acts as a proton donor also during NO reduction. The results are discussed in the framework of a structural model that identifies possible candidates for the proton donor as well as for the proton-transfer pathway.

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