Molecular basis for specificity of the extracytoplasmic thioredoxin ResA.
ResA, an extracytoplasmic thioredoxin from Bacillus subtilis, acts in cytochrome c maturation by reducing the disulfide bond present in apocytochromes prior to covalent attachment of heme. This reaction is (and has to be) specific, as broad substrate specificity would result in unproductive shortcircuiting with the general oxidizing thioredoxin(s) present in the same compartment. Using mutational analysis and subsequent biochemical and structural characterization of active site variants, we show that reduced ResA displays unusually low reactivity at neutral pH, consistent with the observed high pKa values>8 for both active site cysteines. Residue Glu80 is shown to play a key role in controlling the acid-base properties of the active site. A model in which substrate binding dramatically enhances the reactivity of the active site cysteines is proposed to account for the specificity of the protein. Such a substratemediated activation mechanism is likely to have wide relevance for extracytoplasmic thioredoxins.
Lewin
Allison
A
School of Chemical Sciences and Pharmacy, Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Crow
Allister
A
Oubrie
Arthur
A
Le Brun
Nick E
NE
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism*
D001426:Bacterial Proteins; D013879:Thioredoxins