The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH-redoxin family

摘要

NrdH-redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH-redoxins have a CVQC active site motif and belong to the thioredoxin-fold protein family. As for other thioredoxin-fold proteins, the pKa of the nucleophilic cysteine of NrdH-redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pKa value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH-redoxins were determined, but structural insights explaining the relatively low pKa remained elusive. We subjected C. glutamicum NrdH-redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pKa of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N-terminal of the active site further lowers the cysteine pKa. However, site-directed mutagenesis data show that the major contribution to the lowering of the cysteine pKa comes from the positive charge of the lysine and not from the additional Lys-Cys hydrogen bond. In 12% of the NrdH-redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pKa. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N-terminally of the active site dynamically regulate the pKa of the nucleophilic cysteine in NrdH-redoxins.