TY - JOUR
T1 - Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase
AU - Cui, Chang
AU - Song, David Y.
AU - Drennan, Catherine L.
AU - Stubbe, Jo Anne
AU - Nocera, Daniel G.
N1 - Funding Information:
This work was supported by the National Institutes of Health Grant GM047274 (D.G.N.) and GM126982 (C.L.D.). C.L.D. is an HHMI Investigator.
Funding Information:
We are grateful to Dr. Yangzhong Qin for helpful discussions with regard to the design of the power dependence of the photochemical activity assay and to Dr. Rui Sun for preparing tricarbonyl(1,10-phenanthroline)(4-bromomethylpyridine) rhenium(I) hexafluorophosphate ([Re]-Br). This work was supported by the National Institutes of Health Grant GM047274 (D.G.N.) and GM126982 (C.L.D.). C.L.D. is an HHMI Investigator.
Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/3/8
Y1 - 2023/3/8
N2 - 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 (C439•), and the β subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[β] ↔ W48?[β] ↔ Y356[β] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[β] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/β interface. An E52[β] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[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, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[β], R331[α], E326[α], and E326[α′] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.
AB - 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 (C439•), and the β subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[β] ↔ W48?[β] ↔ Y356[β] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[β] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/β interface. An E52[β] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[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, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[β], R331[α], E326[α], and E326[α′] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.
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U2 - 10.1021/jacs.2c11483
DO - 10.1021/jacs.2c11483
M3 - Article
C2 - 36812162
AN - SCOPUS:85148771656
SN - 0002-7863
VL - 145
SP - 5145
EP - 5154
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 9
ER -