Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production

Abstract

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.

Figure 1

The spectrum of His-tagged, recombinant C. reinhardtii nitrite reductase. Nitrite reductase was present at a concentration of 3.13 µM in 10 mM potassium phosphate buffer, pH 7.7. The spectrum was recorded at ambient temperature in a 1.0 cm optical pathlength cuvette.

Figure 2

X-band EPR spectra of oxidized, cyanide-bound and dithionite-reduced cyanide-bound wild type C. reinhardtii nitrite reductase. The cyanide-bound sample was prepared by treating the oxidized sample with a 10-fold molar excess of KCN in 250 mM potassium phosphate buffer at pH 7.8. The cyanide-bound nitrite reductase sample was anaerobically reduced using a 10-fold molar excess of sodium dithionite. EPR spectra were recorded at 15 K with a microwave frequency of 9.604 GHz and a modulation amplitude of 6.4 Gauss. A microwave power of 0.02 mW, 2 mW and 20 mW was applied to the dithionite-reduced cyanide-bound, oxidized and cyanide-bound C. reinhardtii nitrite reductase samples, respectively.

Figure 3

Spectral perturbations arising from complex formation between C. reinhardtii nitrite reductase and nitrite. C. reinhardtii nitrite reductase was present at a concentration of 3.0 µM in 250 mM potassium phosphate buffer (pH 7.7). Sodium nitrite was present at a concentration of 32 µM. The difference spectrum shown is that of the complex minus that of nitrite reductase alone. All spectra were measured at ambient temperature in a 1.0 cm optical pathlength cuvette.

Figure 4

Spectral perturbations arising from complex formation between C. reinhardtii nitrite reductase and ferredoxin. C. reinhardtii nitrite reductase was present at a concentration of 3.13 µM and C. reinhardtii ferredoxin 1 was present at a concentration of 10.0 µM in 10 mM potassium phosphate buffer (pH 7.7). The difference spectrum shown is that of the mixture of the two proteins, from which has been subtracted the sum of the spectra of the two separate proteins. All spectra were measured at ambient temperature in a 1.0 cm optical pathlength cuvette.

Figure 5

The effect of ionic strength on the activity of C. reinhardtii nitrite reductase. The ferredoxin-dependent (open squares) and methyl viologen-dependent activities (open circles) were assayed as described in the Methods section except that the concentration of potassium phosphate buffer present in the assay mixture was varied as indicated on the x-axis.

Figure 6

The effect of N-acetylsuccinimide on the activity of C. reinhardtii nitrite reductase. Incubation of the enzyme with N-acetylsuccinimide was carried out as described in Ref. . The enzyme concentration during the incubation was approximately 300 µM and the concentration of N-acetylsuccinimide was 3.0 mM. At the indicated times, the incubation mixture was diluted with 250 mM potassium phosphate buffer and activity with either reduced ferredoxin as the electron donor (open squares) or reduced methyl viologen as the electron donor (open circles) was assayed as described in the Methods section. The open diamonds represent an experiment in which the incubation with N-acetylsuccinimide is carried out with the 1:1 complex of the C. reinhardtii nitrite reductase with ferredoxin instead of with the enzyme alone.

Figure 7

Hydroxylamine reduction catalyzed by C. reinhardtii and spinach nitrite reductase. The 1.0 ml reaction mixture contained, in 62.5 mM potassium phosphate buffer (pH 7.5), 50 µM C. reinhardtii ferredoxin 1, 0.5 mM NADPH, 1.4 µM FNR, 0.3 µM C. reinhardtii nitrite reductase and either 2.0 mM sodium nitrite (open squares) or 2.0 mM hydroxylamine (open circles). The rates were calculated from the absorbance changes at 340 nm, 10 seconds after the reaction was initiated by the addition of either sodium nitrite or hydroxylamine.

Figure 8

Superposition of the structural models of the backbones of nitrite reductases from spinach (red) and C. reinhardtii (blue). The siroheme and [4Fe-4S] cofactors are shown in black.