Spectroscopic and Kinetic Properties of a Recombinant Form of the Flavin Domain of Spinach NADH:Nitrate Reductase

Abstract

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein inEscherichia coli.The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5′-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated 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 form 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 reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH:ferricyanide reductase activity (Vmax= 84 μmol NADH consumed/min/nmol FAD) withKm’s of 17 and 34 μMfor NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5. A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (Vmax= 35 μmol NHDH consumed/min/nmol FAD andKm= 22 μM). NAD+was demonstrated to be a competitive inhibitor (Ki= 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki= 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochromeb5directly (Vmax= 1.2 μmol NADH consumed/min/nmol FAD,Km(cyt.b5) = 6 μM), supporting the FAD →b557→ Mo electron transfer sequence in spinach nitrate reductase.