Defining the proton entry point in the bacterial respiratory nitric-oxide reductase.
The bacterial respiratory nitric-oxide reductase (NOR) is a member of the superfamily of O(2)-reducing, proton-pumping, heme-copper oxidases. Even although nitric oxide reduction is a highly exergonic reaction, NOR is not a proton pump and rather than taking up protons from the cytoplasmic (membrane potential-negative) side of the membrane, like the heme-copper oxidases, NOR derives its substrate protons from the periplasmic (membrane potential-positive) side of the membrane. The molecular details of this non-electrogenic proton transfer are not yet resolved, so in this study we have explored a role in a proposed proton pathway for a conserved surface glutamate (Glu-122) in the catalytic subunit (NorB). The effect of substituting Glu-122 with Ala, Gln, or Asp on a single turnover of the reduced NOR variants with O(2), an alternative and experimentally tractable substrate for NOR, was determined. Electron transfer coupled to proton uptake to the bound O(2) is severely and specifically inhibited in both the E122A and E122Q variants, establishing the importance of a protonatable side chain at this position. In the E122D mutant, proton uptake is retained but it is associated with a significant increase in the observed pK(a) of the group donating protons to the active site. This suggests that Glu-122 is important in defining this proton donor. A second nearby glutamate (Glu-125) is also required for the electron transfer coupled to proton uptake, further emphasizing the importance of this region of NorB in proton transfer. Because Glu-122 is predicted to lie near the periplasmic surface of NOR, the results provide strong experimental evidence that this residue contributes to defining the aperture of a non-electrogenic "E-pathway" that serves to deliver protons from the periplasm to the buried active site in NOR.
Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Ulrika
U
Flock
Faye H
FH
Thorndycroft
Andrey D
AD
Matorin
David J
DJ
Richardson
Nicholas J
NJ
Watmough
Pia
P
Adelroth
D004579:Electron Transport; D008024:Ligands; D008958:Models, Molecular; D010088:Oxidoreductases / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000502:physiology; D011522:Protons
D008024:Ligands; D011522:Protons; D010088:Oxidoreductases; C034747:nitric-oxide reductase