Mechanism and Catalytic Site Atlas

Introduction

The resting state of SiRHP is assumed to contain oxidised cofactors, i.e. Fe(III) in the sirohaem and [4Fe4S]2+, with inorganic phosphate coordinating the sixth position of the sirohaem iron. The first step of the reaction involves reduction of the two cofactors (to Fe(II) and [4Fe4S]+ by transfer of electrons from the FMN in the SiRFP subunits of the complex to the 4Fe4S centre. Electron transfer between the 4Fe4S centre and the sirohaem is facilitated by Cys 483, the side chain thiol of which is a ligand to both. The details of electron transfer from FMN in SiRFP to the 4Fe4S centre are not yet well understood.

Reduction of the cofactors is followed by dissociation of the phosphate. Sulphite now binds to the sirohaem iron as HSO3-. It receives two electrons from the reduced cofactors, takes up a proton from a water molecule, and releases its O3 oxygen as water. Movement of electrons onto the sulphite is promoted by the positively charged residues Arg 83, Arg 153, Lys 215 and Lys 217; these 'pull' negative charge onto the substrate while the electron-rich cofactors 'push' electrons onto it. The oxidised cofactors are now re-reduced by SiRFP, and can then transfer two more electrons to the substrate. Uptake of two protons, probably from water molecules, and release of a second water molecule from the substrate leaves a mono-oxygenated sulphur species. Transfer of another pair of electrons and protons in the same way with loss of a water molecule leaves sulphide, S2-, which takes up a proton and dissociates from the sirohaem as HS- (sulphide is a poor sirohaem ligand). Conformational changes of the positively charged active site residues, particularly Arg 153, occur at specific points during the catalytic cycle; this is important in maintaining interactions with the substrate as the reduction proceeds and oxygen atoms are lost from it.

Reduction of nitrite to ammonia is thought to occur in a similar way, with NO2- initially binding to the sirohaem and coordinated NO and hydroxylamine as possible intermediates. As with sulphite reduction, the exact timing of electron and proton transfers to the substrate remains to be established.