Direct NMR observation of the Cys‐14 thiol proton of reduced Escherichia coli glutaredoxin‐3 supports the presence of an active site thiol‐thiolate hydrogen bond

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Abstract
The active site of Escherichia coli glutaredoxin‐3 (Grx3) consists of two redox active cysteine residues in the sequence ‐C11‐P‐Y‐C14‐H‐. The 1H NMR resonance of the cysteine thiol proton of Cys‐14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys‐11 thiolate, which is shown to have an abnormally low pK a value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys‐11 thiolate.