Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl...

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Autores principales:	Ane B Tomter, Giorgio Zoppellaro, Caleb B Bell, Anne-Laure Barra, Niels H Andersen, Edward I Solomon, K Kristoffer Andersson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/154cc23f8ee94fbca899e78116e34c3f
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spelling	oai:doaj.org-article:154cc23f8ee94fbca899e78116e34c3f2021-11-18T07:25:09ZSpectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.1932-620310.1371/journal.pone.0033436https://doaj.org/article/154cc23f8ee94fbca899e78116e34c3f2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22432022/?tool=EBIhttps://doaj.org/toc/1932-6203Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.Ane B TomterGiorgio ZoppellaroCaleb B BellAnne-Laure BarraNiels H AndersenEdward I SolomonK Kristoffer AnderssonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 3, p e33436 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ane B Tomter Giorgio Zoppellaro Caleb B Bell Anne-Laure Barra Niels H Andersen Edward I Solomon K Kristoffer Andersson Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.
description	Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.
format	article
author	Ane B Tomter Giorgio Zoppellaro Caleb B Bell Anne-Laure Barra Niels H Andersen Edward I Solomon K Kristoffer Andersson
author_facet	Ane B Tomter Giorgio Zoppellaro Caleb B Bell Anne-Laure Barra Niels H Andersen Edward I Solomon K Kristoffer Andersson
author_sort	Ane B Tomter
title	Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.
title_short	Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.
title_full	Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.
title_fullStr	Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.
title_full_unstemmed	Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.
title_sort	spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in bacillus cereus ribonucleotide reductase r2 protein.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/154cc23f8ee94fbca899e78116e34c3f
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Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

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