1yfd: Difference between revisions

Revision as of 17:04, 21 February 2008

Crystal structure of the Y122H mutant of ribonucleotide reductase R2 protein from E. coli

OverviewOverview

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.

About this StructureAbout this Structure

1YFD is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as Ribonucleoside-diphosphate reductase, with EC number 1.17.4.1 Full crystallographic information is available from OCA.

ReferenceReference

A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies., Kolberg M, Logan DT, Bleifuss G, Potsch S, Sjoberg BM, Graslund A, Lubitz W, Lassmann G, Lendzian F, J Biol Chem. 2005 Mar 25;280(12):11233-46. Epub 2005 Jan 5. PMID:15634667

Page seeded by OCA on Thu Feb 21 16:04:47 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1yfd.gif|left|200px]]<br /><applet load="1yfd" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1yfd.gif|left|200px]]<br /><applet load="1yfd" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1yfd, resolution 1.90&Aring;" />
 '''Crystal structure of the Y122H mutant of ribonucleotide reductase R2 protein from E. coli'''<br />
 ==Overview==
-The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs, to a structurally related family of oxygen bridged diiron proteins. In, wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen, by the diferrous iron center generates a radical on a nearby tyrosine, residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we, characterize the mutant E. coli protein R2-Y122H, where the radical site, is substituted with a histidine residue. The x-ray structure verifies the, mutation. R2-Y122H contains a novel stable paramagnetic center which we, name H, and which we have previously proposed to be a diferric iron center, with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a, detailed characterization of center H, using 1H/2H -14N/15N- and, 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in, the iron reconstitution reaction of R2. Specific deuterium labeling of, phenylalanine residues reveals that the radical results from a, phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of, the iron site, and generation of a phenyl radical requires a very high, oxidation potential, we propose that in Y122H residue Phe208 is, hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and, further oxidized to a phenoxyl radical, which is coordinated to Fe1. This, work demonstrates that small structural changes can redirect the, reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as, observed in the structurally similar methane monoxygenase, and beyond, to, formation of a stable iron-coordinated radical.
+The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.
 ==About this Structure==
-YFD is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with HG and F2O as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Ribonucleoside-diphosphate_reductase Ribonucleoside-diphosphate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.17.4.1 1.17.4.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1YFD OCA].
+YFD is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=HG:'>HG</scene> and <scene name='pdbligand=F2O:'>F2O</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Ribonucleoside-diphosphate_reductase Ribonucleoside-diphosphate reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.17.4.1 1.17.4.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1YFD OCA].
 ==Reference==
@@ Line 19: / Line 19: @@
 [[Category: Lassmann, G.]]
 [[Category: Lendzian, F.]]
-[[Category: Logan, D.T.]]
+[[Category: Logan, D T.]]
 [[Category: Lubitz, W.]]
 [[Category: Poetsch, S.]]
-[[Category: Sjoeberg, B.M.]]
+[[Category: Sjoeberg, B M.]]
 [[Category: F2O]]
 [[Category: HG]]
 [[Category: di-iron center]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 06:44:06 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:04:47 2008''