Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N2-fixing cyanobacterium Cyanothece sp. PCC 8801

The assimilatory nitrate reductase (NarB) of N2-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (Km1, ∼1 μM; Km2, ∼270 μM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe–4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1 mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site.

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► The NarB of Cyanothece sp. PCC 8801 contains only single Mo-catalytic center but shows dual-affinity enzyme kinetics.
► 1 mM azide transforms the kinetics of the NarB from 2 kinetic regimes to hyperbolic kinetics.
► The NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site.