PARENT SESSION

Symposium S2C Carbon and nitrogen interactions
Monday August 30th, 2004 2:40 PM-4:20 PM Room 510A
Chair: Steve Huber
Co-Chair: Graham Noctor

Structural, mechanistic and spectroscopic studies of spinach nitrite reductase. Masakazu Hirasawa¹, Sofya Kuznetsova², Pierre Setif², Tony Mattioli², Sung-Kun Kim¹, James Allen³, David Knaff^*,1, ¹ Department of Chemistry and Biochemistry, Lubbock, Texas, U.S.A.² Service de Bioenergetique and CNRS URA 2096, Gif-sur-Yvette, France³ Department of Chemistry and Biochemistry, Tempe, Arizona, U.S.A.

ABSTRACT- The nitrite reductases of oxygenic phototrophs are soluble, monomeric proteins with molecular masses of ca. 63 kDa that use reduced ferredoxin as the electron donor for the 6-electron reduction of nitrite to ammonia. In photosynthetic eukaryotes nitrite reductase are encoded by nuclear genes and are located in the chloroplast stromal space. The enzyme contains only a single binding site for reduced ferredoxin and so electrons enter the enzyme one-at-a-time. As no intermediates are released from the enzyme's active site during the conversion of nitrite to ammonia, the enzyme must accumulate six electrons before ammonia can be released at the end of the catalytic cycle. The ammonia produced by the nitrite reductase-catalyzed reaction is incoroprated into glutamine and represents the start of a major pathway for the assimilation of nitrogen into organic compounds. Ferredoxin-dependent nitrite reductases contain two prosthetic groups, a siroheme and a [4Fe-4S] cluster, with redox properties that have been extensively studied in our laboratory. Before our current work, no tertiary structures were available for any ferredoxin-dependent nitrite reductase. We have used x-ray crystallography to charcaterize the active site of spinach nitrite reductase and have mapped the prosthetic groups at the active site, which have been shown to be coupled by a bridging sulfur atom, and progress is being made towards a complete structure. We have used electron paramagnetic and resonance Raman spectroscopies to examine the oxidation and spin states of the prosthetic groups in the resting enzyme and in enzyme complexes with two likely intermediates formed during the reduction of nitrite to ammonia, i.e., NO and hydroxylamine. We have also used stopped-flow techniques to measure the kinetics of substrate binding to the enzyme. Chemical modification studies have suggested that a tryptophanresidue, located at or near the active site of nitrite reductase. We will report the results of site-directed mutagenesis studies in which each of the eight tryptophan residues in spinach nitrite reductase has beenrepalced by a variety of aromatic and non-aromatic residues to test whether a trtyptophan residue plays an essential role in the cataylytic mechanism of the enzyme.

KEY WORDS: three-dimensional structure, essential tryptophans, nitrite reductase, reaction intermediates