EXPRESSION OF SPINACH NITRITE REDUCTASE IN ESCHERICHIA-COLI - SITE-DIRECTED MUTAGENESIS OF PREDICTED ACTIVE-SITE AMINO-ACIDS

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) in Escherichia coli was 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 beta-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 the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A(278)/A(386) = 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 of E. coli-expressed NiR was 100 U/mg and the K-m determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. 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 Gly(513) --> Ala NiR mutant had a K-m of 1.0 mM for nitrite and an altered absorption spectrum suggesting the chemical environment of the siroheme has been altered. (C) 1995 Academic Press, Inc.