SULFHYDRYL CHEMISTRY DETECTS 3 CONFORMATIONS OF THE LIPID-BINDING REGION OF ESCHERICHIA-COLI PYRUVATE OXIDASE

Site-specific disulfide cross-linking experiments detected a conformat ional change within the C-terminal segment of Escherichia call pyruvat e oxidase (PoxB), a lipid activated homotetrameric enzyme, upon substr ate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995) J. Biol. Chem. 270, 7896-7901]. The C-terminal lipid binding regions were cross-linke d only in the presence of the substrate, pyruvate, and the thiamine py rophosphate cofactor, indicating close proximity of a pair of C termin i. We have now systematically substituted cysteine at 18 additional am ino acid positions within the C-terminal region to obtain a panel of 2 1 proteins each having a single residue changed to cysteine. These pro teins have been studied by disulfide cross-linking and by accessibilit y of the cysteine side chain to a variety of sulfhydryl agents. In the absence of pyruvate, the cysteine residues of the modified PoxB prote ins failed to form disulfide bonds, generally failed to react with a l arge and rigid hydrophilic sulfhydryl reagent, ido-4'-[(iodoacetyl)ami no]stilbene-2,2'-disulfonic acid (IASD), and in some cases reacted wea kly with a smaller more hydrophobic reagent, N-ethylmaleimide. Therefo re, in this conformation, the C termini appear fixed in a rigid enviro nment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal f rom the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a stru cture having a high degree of conformational freedom. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with t he membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles. For most of the proteins, si milar extents of disulfide formation were seen upon addition of an oxi dizing agent in the presence or absence of lipid activators. An except ion was PoxB D560C which was much more readily cross-linked in the pre sence of lipid. Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found. The behavior of these proteins provides strong support for the model in which two C te rmini associate to form the functional lipid binding domain. These dat a are discussed in terms of three distinct PoxB conformers and the kno wn crystal structure of a highly related protein.