Abstract
Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular redox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixed-disulfide intermediate that occurs under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pKa titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements.
Highlights
Thioredoxin (Trx) and glutaredoxin (Grx) are small ubiquitous proteins acting as cysteine disulfide oxidoreductases in the cell and belong to the thioredoxin superfamily [1,2,3]
Different subclasses of the thioredoxin superfamily have nucleophilic cysteine pKa values that correspond to their biological function
The pH-titration curves for wild type and T8A BC3987 in Figure 1 are similar in the pH interval 6.5–9, indicating that the nucleophilic Cys12 pKa value is not affected by the T8A mutation
Summary
Thioredoxin (Trx) and glutaredoxin (Grx) are small ubiquitous proteins acting as cysteine disulfide oxidoreductases in the cell and belong to the thioredoxin superfamily [1,2,3]. Because no structure of reduced BC3987 was obtained, we have modeled the reduced active site using quantum mechanical/molecular mechanical (QM/MM) calculations and rationalized the experimentally observed and unusually low pKa value of the C-terminal buried cysteine through hydrogen bonding to the conserved Thr8 residue.
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