2XOF

Ribonucleotide reductase Y122NO2Y modified R2 subunit of E. coli

Summary for 2XOF

• Entry DOI: 10.2210/pdb2xof/pdb
• Related: 1AV8 1BIQ 1JPR 1JQC 1MRR 1MXR 1PFR 1PIM 1PIU 1PIY 1PIZ 1PJ0 1PJ1 1PM2 1R1R 1R65 1RIB 1RNR 1RSR 1RSV 1XIK 1YFD 2ALX 2AV8 2R1R 2X0X 2XAK 2XAP 2XAV 2XAW 2XAX 2XAY 2XAZ 2XO4 2XO5 3R1R 4R1R 5R1R 6R1R 7R1R
• Descriptor: RIBONUCLEOSIDE-DIPHOSPHATE REDUCTASE 1 SUBUNIT BETA, MU-OXO-DIIRON (3 entities in total)
• Functional Keywords: oxidoreductase, radical storage, dna replication, allosteric enzyme
• Biological source: ESCHERICHIA COLI
• Total number of polymer chains: 2
• Total formula weight: 87199.09

Authors

Yokoyama, K.,Uhlin, U.,Stubbe, J. (deposition date: 2010-08-15, release date: 2010-08-25, Last modification date: 2023-12-20)

Primary citation

Yokoyama, K.,Uhlin, U.,Stubbe, J.
A Hot Oxidant, 3-No(2)Y(122) Radical, Unmasks Conformational Gating in Ribonucleotide Reductase.
J.Am.Chem.Soc., 132:15368-, 2010

PubMed Abstract: Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(•)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•)→W(48)→Y(356) within β to Y(731)→Y(730)→C(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(•)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-β2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(•) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(•)/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(•) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(•) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(•) is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-β2, a mechanism for PCET uncoupling in NO(2)Y(•)-RNR is proposed.

PubMed: 20929229
DOI: 10.1021/JA1069344

Experimental method

X-RAY DIFFRACTION (2.2 Å)