Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase
Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 5′-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essenti...
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| Format: | Article |
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American Chemical Society
2013
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| Online Access: | http://hdl.handle.net/1721.1/82535 https://orcid.org/0000-0001-8076-4489 |
| _version_ | 1826209966289059840 |
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| author | Minnihan, Ellen Catherine Seyedsayamdost, Mohammad R. Stubbe, JoAnne |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Minnihan, Ellen Catherine Seyedsayamdost, Mohammad R. Stubbe, JoAnne |
| author_sort | Minnihan, Ellen Catherine |
| collection | MIT |
| description | Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 5′-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y[subscript 122]•) cofactor which, in the presence of S and E, oxidizes C[subscript 439] in α to a thiyl radical, C[subscript 439]•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y[subscript 122]• --> W[subscript 48 --> Y[subscript 356] in β2 to Y[subscript 731] --> Y[subscript 730] --> C[subscript 439] in α2. 3-Aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH[subscript 2]Y•) has been examined by stopped-flow (SF) UV−vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y[subscript 356]F-β2:Y[subscript 731]NH[subscript 2]Y-α2, Y[subscript 356]F-β2:Y[subscript 730]NH[subscript 2]Y-α2, and wt-β2:Y[subscript 731]F/Y[subscript 730]NH[subscript 2]Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH[subscript 2]Y• was detected. To study off-pathway oxidation, Y[subscript 413], located 5 Å from Y[subscript 730] and Y[subscript 731] but not implicated in long-range oxidation, was examined. Evidence for NH[subscript 2]Y[subscript 413]• was sought in three complexes: wt-β2:Y[subscript 413]NH[subscript 2]Y-α2 (a), wt-β2:Y[subscript 731]F/Y[subscript 413]NH[subscript 2]Y-α2 (b), and Y[subscript 356]F-β2:Y[subscript 413]NH[subscript 2]Y-α2 (c). With (a), NH[subscript 2]Y• was formed with a rate constant that was 25−30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH[subscript 2]Y• formation was 0.2−0.3% of that observed at 731 and 730, and with (c), no NH[subscript 2]Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C[subscript 439]. |
| first_indexed | 2024-09-23T14:37:37Z |
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| id | mit-1721.1/82535 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:37:37Z |
| publishDate | 2013 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/825352022-10-01T21:51:48Z Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase Minnihan, Ellen Catherine Seyedsayamdost, Mohammad R. Stubbe, JoAnne Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Minnihan, Ellen Catherine Seyedsayamdost, Mohammad R. Stubbe, JoAnne Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 5′-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y[subscript 122]•) cofactor which, in the presence of S and E, oxidizes C[subscript 439] in α to a thiyl radical, C[subscript 439]•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y[subscript 122]• --> W[subscript 48 --> Y[subscript 356] in β2 to Y[subscript 731] --> Y[subscript 730] --> C[subscript 439] in α2. 3-Aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH[subscript 2]Y•) has been examined by stopped-flow (SF) UV−vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y[subscript 356]F-β2:Y[subscript 731]NH[subscript 2]Y-α2, Y[subscript 356]F-β2:Y[subscript 730]NH[subscript 2]Y-α2, and wt-β2:Y[subscript 731]F/Y[subscript 730]NH[subscript 2]Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH[subscript 2]Y• was detected. To study off-pathway oxidation, Y[subscript 413], located 5 Å from Y[subscript 730] and Y[subscript 731] but not implicated in long-range oxidation, was examined. Evidence for NH[subscript 2]Y[subscript 413]• was sought in three complexes: wt-β2:Y[subscript 413]NH[subscript 2]Y-α2 (a), wt-β2:Y[subscript 731]F/Y[subscript 413]NH[subscript 2]Y-α2 (b), and Y[subscript 356]F-β2:Y[subscript 413]NH[subscript 2]Y-α2 (c). With (a), NH[subscript 2]Y• was formed with a rate constant that was 25−30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH[subscript 2]Y• formation was 0.2−0.3% of that observed at 731 and 730, and with (c), no NH[subscript 2]Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C[subscript 439]. National Institutes of Health (U.S.) (Grant GM29595) David H. Koch Institute for Integrative Cancer Research at MIT (Koch Graduate Fellowship) 2013-11-21T21:34:14Z 2013-11-21T21:34:14Z 2009-12 2009-11 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/82535 Minnihan, Ellen C., Mohammad R. Seyedsayamdost, and JoAnne Stubbe. "Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase.” Biochemistry 48, no. 51 (December 29, 2009): 12125-12132. https://orcid.org/0000-0001-8076-4489 en_US http://dx.doi.org/10.1021/bi901439w Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society PMC |
| spellingShingle | Minnihan, Ellen Catherine Seyedsayamdost, Mohammad R. Stubbe, JoAnne Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase |
| title | Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase |
| title_full | Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase |
| title_fullStr | Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase |
| title_full_unstemmed | Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase |
| title_short | Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase |
| title_sort | use of 3 aminotyrosine to examine the pathway dependence of radical propagation in escherichia coli ribonucleotide reductase |
| url | http://hdl.handle.net/1721.1/82535 https://orcid.org/0000-0001-8076-4489 |
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