Nitrate Reductase Biochemistry Comes of Age

NR (EC 1.6.6.1-3) was first isolated and characterized more than 40 years ago, and each decade of study of this key enzyme of nitrate assimilation has been associated with a new understanding of its structure and function. Briefly, NR is a homodimeric enzyme (native form = A,) with each subunit containing a 100-kD polypeptide and one each of molybdate, Mo-pterin, Fe, heme, and FAD (Redinbaugh and Campbell, 1985). NR has two active sites, one where NADH donates electrons to FAD to begin the transport of electrons via the heme-Fe to the Mo / Mo-pterin in the second active site, where nitrate is reduced to nitrite. NR has two-site, ping-pong, steady-state kinetics, where the enzyme “pings and pongs” between oxidized and reduced forms, as NADH/NADC bind at the electron donor active site and nitrate/nitrite bind at the electron acceptor site. The most recent advances have resulted from the cloning of the NR gene (Campbell and Kinghorn, 1990; Solomonson and Barber, 1990; Rouze and Caboche, 1992). The deduced amino acid sequence of higher-plant NR showed that it contained about 900 residues with a predicted molecular size of approximately 100 kD. Although this size is a bit smaller than the NR polypeptide appears on SDS-PAGE gels (110-115 kD; Redinbaugh and Campbell, 1985), this may be explained by the runs of acidic residues near the N terminus of well-characterized and sequenced NR forms such as squash (Hyde et al., 1991). In this general review, I will focus on recent advances in NR biochemistry. I last reviewed this topic in a general way in 1988 (Campbell, 1988). Reviews by Solomonson and Barber (1990), Rouze and Caboche (1992), and Crawford (1995) provide more detailed accounts of various aspects of this topic than will be presented here. Recently, Kaiser and Huber (1994) reviewed posttranslational regulation of NR in an Update.