Proposed reaction mechanism for the reduction of the cysteine-sulfinic acid of Tsa1 by yeast Srx1. The reaction proceeds from a sulfinic acid through sulfinic phosphoryl ester and thiosulfinate intermediates (Biteau et al., 2003). The position of ATP hydrolysis and the identity of the exogenous thiol (R-SH) were proposed to be between the β-γ phosphate and thioredoxin, respectively.

Sequence alignment of representative sulfiredoxins. The homology of the proteins to human Srx decreases down the alignment. The Srxs from mouse, Drosophila, Arabidopsis, Nostoc species PCC7120 (a cyano-bacterium), and S. cerevisiae show 91%, 60%, 43%, 41%, 33% sequence identity to hSrx, respectively. The secondary structural elements for human Srx are shown above the alignment: α, α-helices; β, β-strands; η, 3₁₀ helices (See Figure 3 and section 5.1). The residues highlighted by the red background and white lettering are strictly conserved. Residues that are either conserved in the majority of the proteins or have conservative substitutions are boxed in blue and colored red. The black dots above the alignment indicate every tenth residue of human Srx.

Crystal structure of human Srx. (A) Native enzyme in complex with phosphate. The α-helices and β-strands within the novel fold of Srx are numbered consecutively based on the primary sequence. Residues of the signature sequence of Srx are highlighted. For clarity residues 29-36 and the 3₁₀-helicies are not shown. Atom color scheme: green, carbon atoms for Srx; yellow, carbon atoms for ATP; red, oxygen; blue, nitrogen; magenta, phosphorous; orange, sulfur. (B) Model of ATP bound to Srx. The ATP:Srx complex is based on the crystal structure of the ADP complex. Putative hydrogen bonding interactions are indicated by dashed yellow lines. (C) Surface representation of Srx in complex with ATP. Strictly and semi-conserved residues are indicated in black and red, respectively. Carbon atoms for the Srx surface are shown in gray.

Proposed binding mode of ATP and the first step in the Srx reaction. Putative hydrogen bonding interactions are indicated by dashed lines. The nucleotide and sugar structures of the ATP molecule are abbreviated by the letter A.

Inaccessibility of the hyperoxidized active site of human PrxII. Hyperoxidized 2-Cys Prxs form stabilized decamers. Each monomer is colored differently. A close-up view of one PrxII active sites (blue surface and ribbon) illustrates the difficulty Srx has in gaining access to the Cys51-S_PO₂^- moiety (Csd51) which is involved in a hydrogen bonding interaction with Arg127. The Prx active site is occluded primarily by residues of the YF motif within the C-terminal α-helix of the adjacent Prx molecule (magenta surface and ribbon). This latter structural feature also interacts with the GGLG motif, another conserved region found primarily in Prxs sensitive to hyperoxidation.

Model for the Srx:Prx interaction. The top panel indicates the edge-on view of four subunits of a Prx decamer. The active sites of the middle Prx dimer (green) are indicated in cyan. The two-fold axis is indicated by the red dyad symbol. Adjacent subunits in the Prx decamer are shown as the non-filled circles. The bottom panel illustrates the putative binding mode of Srx (blue) to the circumference of the decamer and the central Prx dimer. The Srx molecules most likely also adopt a two-fold relationship indicated by the plus signs. It is possible that the Srx molecules dock more to the side of the Prx decamer maintaining the symmetry relationship.

Comparison of the proposed reaction mechanisms for Srx. Path 1 represents the original mechanism as proposed for yeast Srx1. Path 2 incorporates potential modifications to the reaction pathway based on biochemical experiments with mammalian Srxs in both the wild-type and mutant forms. Step 1 involves the formation of the sulfinic phosphoryl ester intermediate. In step 2 of the reaction, the addition of a thiol group leads to the formation of alternative thiosulfinate intermediates. Breakdown of the latter intermediate occurs in steps 3 and following via reduction by additional thiol-disulfide exchange reactions.