SPECTROSCOPIC AND KINETIC-PROPERTIES OF A RECOMBINANT FORM OF THE FLAVIN DOMAIN OF SPINACH NADH-NITRATE REDUCTASE

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate r eductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, wh ich was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, aff inity chromatography on 5'-ADP-agarose and FPLC gel filtration, The pu rified domain exhibited a molecular weight of approximately 30 kDa, es timated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD, The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized f orm while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively, The purified domain showed immunological cross-reactivity with anti-spinach nitrate reduct ase polyclonal antibodies while both N-terminal and internal amino aci d sequencing of isolated peptides confirmed the fidelity of the domain 's primary sequence, The protein retained NADH:ferricyanide reductase activity (V-max = 84 mu mol NADH consumed/min/nmol FAD) with K-m's of 17 and 34 mu M for NADH and ferricyanide, respectively, with a pH opti mum of approximately 6.5, A variety of NADH-analogs could also functio n as electron donors, though with decreased efficiency, the most effec tive being reduced nicotinamide hypoxanthine dinucleotide (V-max = 35 mu mol NHDH consumed/min/nmol FAD and K-m = 22 mu M). NAD(+) was demon strated to be a competitive inhibitor (K-i = 1.9 mM) while analysis of inhibition by a variety of NAD(+)-analogs indicated the most efficien t inhibitor to be ADP (K-i = 0.2 mM), with analogs devoid of either th e phosphate, ribose, or adenine moieties proving to be markedly less-e fficient inhibitors, The isolated domain was also capable of reducing cytochrome b(5) directly (V-max = 1.2 mu mol NADH consumed/min/nmol FA D, K-m (cyt, b(5)) = 6 mu M), supporting the FAD --> b(557) --> Mo ele ctron transfer sequence in spinach nitrate reductase. (C) 1996 Academi c Press, Inc.