Local and Global Effects of Metal Binding within the Small Subunit of Ribonucleotide Reductase
- 18 February 2005
- journal article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 127 (10) , 3613-3623
- https://doi.org/10.1021/ja0491937
Abstract
Each beta-protomer of the small betabeta subunit of Escherichia coli ribonucleotide reductase (R2) contains a binuclear iron cluster with inequivalent binding sites: Fe(A) and Fe(B). In anaerobic Fe(II) titrations of apoprotein under standard buffer conditions, we show that the majority of the protein binds only one Fe(II) atom per betabeta subunit. Additional iron occupation can be achieved upon exposure to O2 or in high glycerol buffers. The differential binding affinity of the A- and B-sites allows us to produce heterobinuclear Mn(II)Fe(II) and novel Mn(III)Fe(III) clusters within a single beta-protomer of R2. The oxidized species are produced with H2O2 addition. We demonstrate that no significant exchange of metal occurs between the A- and B-sites, and thus the binding of the first metal is under kinetic control, as has been suggested previously. The binding of first Fe(II) atom to the active site in a beta-protomer (betaI) induces a global protein conformational change that inhibits access of metal to the active site in the other beta-protomer (betaII). The binding of the same Fe(II) atom also induces a local effect at the active site in betaI-protomer, which lowers the affinity for metal in the A-site. The mixed metal FeMn species are quantitatively characterized with electron paramagnetic resonance spectroscopy. The previously reported catalase activity of Mn2(II)R2 is shown not to be associated with Mn.Keywords
This publication has 33 references indexed in Scilit:
- Mechanistic Implications for the Formation of the Diiron Cluster in Ribonucleotide Reductase Provided by Quantitative EPR SpectroscopyJournal of the American Chemical Society, 2003
- High-Frequency EPR Study of the Ferrous Ion in the Reduced Rubredoxin Model [Fe(SPh)4]2-Inorganic Chemistry, 1999
- The Crystal Structure of an Azide Complex of the Diferrous R2 Subunit of Ribonucleotide Reductase Displays a Novel Carboxylate Shift with Important Mechanistic Implications for Diiron-Catalyzed Oxygen ActivationJournal of the American Chemical Society, 1999
- Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron siteStructure, 1996
- Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. Coli Ribonucleotide Reductase: 1. Moessbauer Characterization of the Diferric Radical PrecursorJournal of the American Chemical Society, 1994
- Azide binding to the diferrous clusters of the R2 protein of ribonucleotide reductase from Escherichia coliJournal of the American Chemical Society, 1993
- Structure and Function of the Escherichia coli Ribonucleotide Reductase Protein R2Journal of Molecular Biology, 1993
- Mechanism of Assembly of the Tyrosyl Radical-Dinuclear Iron Cluster Cofactor of Ribonucleotide ReductaseScience, 1991
- Novel diferric radical intermediate responsible for tyrosyl radical formation in assembly of the cofactor of ribonucleotide reductaseJournal of the American Chemical Society, 1991
- Electronic structure and bonding of the blue copper site in plastocyaninJournal of the American Chemical Society, 1985