OXIDIZED AND REDUCED NITRITE REDUCTASE WITH AND WITHOUT NITRITE BOUND Elinor T. Adman, Michael E.P.Murphy, Stewart Turley, Mutsuko Kukimoto, and Makoto Nishiyama. Dept of Biological Structure, University of Washington, Seattle WA, 98195-7420 and Dept of Biotechnology, University of Tokyo, Yayoi 1-1-1 Bunkyo-ku, Tokyo 113 JAPAN.
Dissimilatory nitrite reductase is a trimeric copper-containing protein in Alcaligenes faecalis and Achromobacter cycloclastes. NO is the immediate product and is an intermediate in the dissimilatory denitrification pathway, in which nitrate is completely reduced to N2. Data from crystals of Alcaligenes faecalis NIR at -160deg. C in its oxidized and reduced forms, and with nitrite soaked into crystals, show that the reduced form binds ligands much less tightly than the oxidized form. Previouly published results have shown that the electron transfer partner of nitrite reductase, pseudoazurin, donates electrons to NIR via the Type I Cu site. Electrons are then transferred internally to the Type II Cu site, normally liganded by three histidines and a solvent. A more weakly bound ligand in the reduced form suggests that nitrite first replaces the solvent ligand in the oxidized form, modifying the redox potential of the active site Type II copper so that electrons are then transferred from the Type I site.
Four data sets were collected at -160deg. C on an R-axis II image plate using crystals cryoprotected in PEG, glycerol and methanol. Oxidized crystals were soaked in nitrite at room temperature and then cooled. Crystals reduced with ascorbate were cooled to -40deg. C before nitrite was added. The average cell dimensions were a=61.84 Å, b=102.6 Å, c=146.2 Å, space group P212121, 5% smaller in volume than the room temperature cell. Difference maps using phases from a rigid body refinement of PDB coordinates AFN2 (R=0.29, 10-2 Å) revealed the clearest view of nitrite in the active site when amplitudes from oxidized + nitrite crystals minus amplitudes from reduced crystals were used.
This work has been supported by NIH grant GM31770 and the Medical Research Council of Canada.