Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b557-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b557-containing fragment, corresponding to residues A542–E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647∗ truncated form resulted in substantial heme domain production as evidenced by the generation of “pink” cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS–PAGE analysis with a molecular mass of approximately 13 kDa. MALDI–TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation–reduction titrations of the heme domain indicated a standard midpoint potential (Eo′) of −118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a KA of 7 μM (μ=0.007) and reconsituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a kcat of 1.4 s−1 and a Km app for cytochrome c of 9 μM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.