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J. Biol. Chem., Vol. 282, Issue 9, 6347-6355, March 2, 2007

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pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase^*

Frida Jacobson, Arthur Pistorius¹, Daniel Farkas^¶, Willem De Grip², Örjan Hansson^¶, Lennart Sjölin^||, and Richard Neutze³

From the Department of Chemical and Biological Engineering, Chalmers University of Technology, Box 462, SE-40530 Göteborg, Sweden, Department of Biochemistry, UMC-286, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands, ^¶Biochemistry and Biophysics, Department of Chemistry, Göteborg University, Box 462, SE-40530 Göteborg, Sweden, and ^||Inorganic Chemistry, Department of Chemistry, Göteborg University, SE-41296 Göteborg, Sweden

Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.

Received for publication, June 15, 2006 , and in revised form, November 22, 2006.

The atomic coordinates and structure factors (code 2DWS, 2DY2, and 2DWT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Swedish Natural Science Research Council and Stint. 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.

¹ Supported by the Chemical Council of the Netherlands Organization for Scientific Research (NWO-CW).

² Supported by the Chemical Council of the Netherlands Organization for Scientific Research (NWO-CW) and by European Union Grant LSHG-CT-2004-504601 (to the E-Mep consortium).

³ To whom correspondence should be addressed. E-mail: richard.neutze{at}chembio.chalmers.se; Tel.: 46-31-7733974; Fax: 46-31-7733910.

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