Expression of Spinach Nitrite Reductase inEscherichia coli:Site-Directed Mutagenesis of Predicted Active Site Amino Acids

Abstract

Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]–siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) inEscherichia coliwas developed in order to study the structure–function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a β-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5–1.0% of the total protein.E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS–polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of theE. coli-expressed NiR was identical to authentic spinach NiR with a Soret and α band at 386 and 573 nm, respectively, and aA278/A386= 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite ofE. coli-expressed NiR was 100 U/mg and theKmdetermined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that theE. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state. Site-directed mutations were introduced into amino acids conserved between NiR and the sulfite reductase hemoprotein surrounding the active site to examine structure–function relationships in this enzyme. Replacement of glycines in the active site with progressively larger amino acids resulted in an increasing loss of activity suggesting there is a steric limitation at this position for the formation of the active site. Purified Gly513→ Ala NiR mutant had aKmof 1.0 mMfor nitrite and an altered absorption spectrum suggesting the chemical environment of the siroheme has been altered.