ELIMINATION OF ALL CHARGED RESIDUES IN THE VICINITY OF THE ACTIVE-SITE HELIX OF THE DISULFIDE OXIDOREDUCTASE DSBA - INFLUENCE OF ELECTROSTATIC INTERACTIONS ON STABILITY AND REDOX PROPERTIES

Disulfide oxidoreductases are structurally related proteins that share the thioredoxin fold and a catalytic disulfide bond that is located a t the N terminus of an alpha-helix, The different redox potentials of these enzymes varying from -270 mV for thioredoxin to -125 mV for DsbA have been attributed to the lowered pK(a) values of their nucleophili c, active-site cysteines and the difference in thermodynamic stability between their oxidized and reduced forms (Delta Delta G(ox/red)). The lowered pK(a) of the nucleophilic cysteine thiols was proposed to res ult from favorable interactions with the helix dipole and charged resi dues in their vicinity, In this study, we have eliminated all charged residues in the neighborhood of the active-site disulfide of DsbA from Escherichia coli to analyze their contribution to the physicochemical properties of the protein, We show that the conserved charge network among residues Glu(24), Glu(37), and Lys(58) stabilizes the oxidized f orm of DsbA and thus does not cause the high redox potential of the en zyme, The pK(a) values of the nucleophilic cysteine (Cys(30)) and the redox potentials of the DsbA variants E24Q, E37Q, K58M, E24Q/K58M, E37 Q/K58M, E24Q/E37Q, E24Q/E37Q/K58M, and E24Q/E37Q/E38Q/K58M are similar to those of DsbA wild type, The redox potentials of the variants neit her correlate with the Cys(30) pK(a) values nor with the Delta Delta G (ox/red) values, demonstrating that the relationship between these par ameters is far more complex than previously thought.