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.
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 pKa 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.
Nordstrand
K
K
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Aslund
F
F
Meunier
S
S
Holmgren
A
A
Otting
G
G
Berndt
K D
KD
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D054477:Glutaredoxins; D006860:Hydrogen Bonding; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D001426:Bacterial Proteins; D054477:Glutaredoxins; D011506:Proteins; D011522:Protons; D013438:Sulfhydryl Compounds; D010088:Oxidoreductases; D003545:Cysteine