| Autor: |
Cui C; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States., Song DY; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States., Drennan CL; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States., Stubbe J; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States.; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.; Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States., Nocera DG; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States. |
| Abstrakt: |
Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and β. The active form is an asymmetric αα'ββ' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C 439 •), and the β subunit houses the diferric-tyrosyl radical (Y 122 •) that is essential for C 439 • formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y 122 •[β] ↔ W 48 ?[β] ↔ Y 356 [β] ↔ Y 731 [α] ↔ Y 730 [α] ↔ C 439 [α]. In a recent cryo-EM structure, Y 356 [β] was revealed for the first time and it, along with Y 731 [α], spans the asymmetric α/β interface. An E 52 [β] residue, which is essential for Y 356 oxidation, allows access to the interface and resides at the head of a polar region comprising R 331 [α], E 326 [α], and E 326 [α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y 356 • was photochemically generated using a photosensitizer covalently attached adjacent to Y 356 [β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E 52 [β], R 331 [α], E 326 [α], and E 326 [α'] network plays the essential role of shuttling protons associated with Y 356 oxidation from the interface to bulk solvent. |
