The role of extended Fe4S4 cluster ligands in mediating sulfite reductase hemoprotein activity.

The siroheme-containing subunit from the multimeric hemoflavoprotein NADPH-dependent sulfite reductase (SiR/SiRHP) catalyzes the six electron-reduction of SO32- to S2-. Siroheme is an iron-containing isobacteriochlorin that is found in sulfite and homologous siroheme-containing nitrite reductases. Siroheme does not work alone but is covalently coupled to a Fe4S4 cluster through one of the cluster's ligands. One long-standing hypothesis predicted from this observation is that the environment of one iron-containing cofactor influences the properties of the other. We tested this hypothesis by identifying three amino acids (F437, M444, and T477) that interact with the Fe4S4 cluster and probing the effect of altering them to alanine on the function and structure of the resulting enzymes by use of activity assays, X-ray crystallographic analysis, and EPR spectroscopy. We showed that F437 and M444 gate access for electron transfer to the siroheme-cluster assembly and the direct hydrogen bond between T477 and one of the cluster sulfides is important for determining the geometry of the siroheme active site.

A novel variant of ferredoxin-dependent sulfite reductase having preferred substrate specificity for nitrite in the unicellular red alga Cyanidioschyzon merolae.

Plant NiR (nitrite reductase) and SiR (sulfite reductase) have common structural and functional features. Both enzymes are generally distinguished in terms of substrate specificity for nitrite and sulfite. The genome of Cyanidioschyzon merolae, a unicellular red alga living in acidic hot springs, encodes two SiR homologues, namely CmSiRA and CmSiRB (C. merolae sulfite reductases A and B), but no NiR homologue. The fact that most known SiRs have a low nitrite-reducing activity and that the CmSiRB gene is mapped between the genes for nitrate transporter and nitrate reductase implies that CmSiRB could have a potential to function as a nitrite-reducing enzyme. To verify this hypothesis, we produced a recombinant form of CmSiRB and characterized its enzymatic properties. The enzyme was found to have a significant nitrite-reducing activity, whereas its sulfite-reducing activity was extremely low. As the affinity of CmSiRB for sulfite was higher by 25-fold than that for nitrite, nitrite reduction by CmSiRB was competitively inhibited by sulfite. These results demonstrate that CmSiRB is a unique SiR having a decreased sulfite-reducing activity and an enhanced nitrite-reducing activity. The cellular level of CmSiRB was significantly increased when C. merolae was grown in a nitrate medium. The nitrate-grown C. merolae cells showed a high nitrite uptake from the growth medium, and this consumption was inhibited by sulfite. These combined results indicate that CmSiRB has a significant nitrite-reducing activity and plays a physiological role in nitrate assimilation.