Mutational analysis of the substrate specificity of plant sulfite reductase

Abstract

Plants have ferredoxin-dependent sulfite reductase (SiR: E.C.1.8.7.1) and nitrite reductase (NiR: E.C.1.7.7.1) for assimilation of inorganic sulfur and nitrogen, respectively. These enzymes contain common prosthetic groups, one [4Fe-4S] cluster and one siroheme, and have partially overlapped substrate specificity, both catalyzing a six-electron reduction of sulfite and nitrite to sulfide and ammonia. In fact, SiR shows a strong preference for sulfite (Km: ~5 µM) over nitrite (Km:~0.5 mM). The substrate anions are coordinated to the siroheme iron at its distal side where putative substrate-binding basic residues, Arg-193, Lys-276 and Lys-278 are located. We tried to clarify the structural basis for such substrate discrimination by site-directed mutagenesis of maize SiR. By a heterologous expression of SiR cDNA in E. coli cells, four mutants, R193E, R193A, K276Q and K278N, were obtained as a holo-enzyme with the two prosthetic groups assembled. All mutants lost the sulfite-reduction activity almost completely, and interestingly some mutants showed higher activity for nitrite reduction than wild type. Kinetic analysis demonstrated that affinity to sulfite of R193A and R193E was decreased by less than one-hundredth, while affinity to nitrite of R193A was increased remarkably to an extent similar to that of NiR (Km: ~2 µM). K276Q retained a significant affinity to both sulfite and nitrite, but neither substrate was reduced. These data indicated that the substrate specificity of SiR is drastically changed by substitution of the single basic residue at the distal side of the siroheme.