Publication Date:
1991-07-29
Description:
Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction. The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy. Two novel intermediates have been detected in the reaction. The first exhibits a broad absorption band centered at 565 nanometers. Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex. The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00. When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical. Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122. This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122. Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed. Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Bollinger, J M Jr -- Edmondson, D E -- Huynh, B H -- Filley, J -- Norton, J R -- Stubbe, J -- GM29433/GM/NIGMS NIH HHS/ -- GM29595/GM/NIGMS NIH HHS/ -- GM32187/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 1991 Jul 19;253(5017):292-8.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge 02139.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/1650033" target="_blank"〉PubMed〈/a〉
Keywords:
Binding Sites
;
Electron Spin Resonance Spectroscopy
;
Escherichia coli/enzymology
;
Iron/*metabolism
;
Kinetics
;
Macromolecular Substances
;
Models, Theoretical
;
Oxygen/*metabolism
;
Ribonucleotide Reductases/*metabolism
;
Spectrophotometry, Ultraviolet
;
Time Factors
;
Tyrosine/*metabolism
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
Permalink
