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J. Biol. Chem., Vol. 283, Issue 7, 3839-3845, February 15, 2008

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Defining the Proton Entry Point in the Bacterial Respiratory Nitric-oxide Reductase^*

Ulrika Flock, Faye H. Thorndycroft¹, Andrey D. Matorin, David J. Richardson, Nicholas J. Watmough, and Pia Ädelroth²

From the Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden and Centre for Molecular Structure and Biochemistry, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

The bacterial respiratory nitric-oxide reductase (NOR) is a member of the superfamily of O₂-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 O₂, an alternative and experimentally tractable substrate for NOR, was determined. Electron transfer coupled to proton uptake to the bound O₂ 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.

Received for publication, June 5, 2007 , and in revised form, November 30, 2007.

^* The work in Stockholm was supported in part by a grant (to P.Ä_.) from the Swedish Research Council. The work in Norwich was supported in part by UK Biotechnology and Biological Sciences Research Council Grants B19851 (to D. J. R.) and BBC0077191 (to N. J. W./D. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by a UK Medical Research Council Priority Area studentship.

² A Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation. To whom correspondence should be addressed. Tel.: 46-8-164183; Fax: 46-8-153679; E-mail: piaa{at}dbb.su.se.

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