Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.
Nitrate reductase A (NRA, NarGHI) is expressed in Escherichia coli by growing the bacterium in anaerobic conditions in the presence of nitrate. This enzyme reduces nitrate to nitrite and uses menaquinol (or ubiquinol) as the electron donor. The location of quinones in the enzyme, their number, and their role in the electron transfer mechanism are still controversial. In this work, we have investigated the spectroscopic and thermodynamic properties of a semiquinone (SQ) in membrane samples of overexpressed E. coli nitrate reductase poised in appropriate redox conditions. This semiquinone is highly stabilized with respect to free semiquinone. The g-values determined from the numerical simulation of its Q-band (35 GHz) EPR spectrum are equal to 2.0061, 2.0051, 2.0023. The midpoint potential of the Q/QH(2) couple is about -100 mV, and the SQ stability constant is about 100 at pH 7.5. The semiquinone EPR signal disappears completely upon addition of the quinol binding site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). A semiquinone radical could also be stabilized in preparations where only the NarI membrane subunit is overexpressed in the absence of the NarGH catalytic dimer. Its thermodynamic and spectroscopic properties show only slight variations with those of the wild-type enzyme. The X-band continuous wave (cw) electron nuclear double resonance (ENDOR) spectra of the radicals display similar proton hyperfine coupling patterns in NarGHI and in NarI, showing that they arise from the same semiquinone species bound to a single site located in the NarI membrane subunit. These results are discussed with regard to the location and the potential function of quinones in the enzyme.
Grimaldi
Stéphane
S
Unité de Bioénergétique et Ingénierie des Protéines (UPR9036), Institut de Biologie Structurale et de Microbiologie, CNRS, and Université de Provence, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. grimaldi@ibsm.cnrs-mrs.fr
Lanciano
Pascal
P
Bertrand
Patrick
P
Blasco
Francis
F
Guigliarelli
Bruno
B
D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006912:Hydroxyquinolines / Q000737:chemistry; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D016227:Benzoquinones; D029968:Escherichia coli Proteins; D006912:Hydroxyquinolines; D021122:Protein Subunits; C010494:nonyl-4-hydroxyquinoline-N-oxide; C025232:semiquinone radicals; D009565:Nitrate Reductases; D050901:Nitrate Reductase