Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase
E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y[subscript 122-]β2 reversibly oxidizes C[subscript 439] in the active site of α2. This radica...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2012
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| Online Access: | http://hdl.handle.net/1721.1/72363 |
| _version_ | 1826190167061299200 |
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| author | Yokoyama, Kenichi Uhlin, Ulla Stubbe, JoAnne |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Yokoyama, Kenichi Uhlin, Ulla Stubbe, JoAnne |
| author_sort | Yokoyama, Kenichi |
| collection | MIT |
| description | E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y[subscript 122-]β2 reversibly oxidizes C[subscript 439] in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y[subscript 122] and Y[subscript 356] in β2, Y[subscript 731] and Y[subscript 730] in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO[subscript 2]Y, pK[subscript a] 7.1) has been incorporated in place of Y[subscript 122], Y[subscript 731], and Y[subscript 730] to probe how the protein environment perturbs each pK[subscript a] in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 × 10[subscript −3] that of the wild-type (wt) subunit. The [NO[subscript 2]Y[subscript 730]]-α2 and [NO[subscript 2]Y[subscript 731]]-α2 each exhibit a pK[subscript a] of 7.8−8.0 with E and E/β2. The pK[subscript a] of [NO[subscript 2]Y[subscript 730]]-α2 is elevated to 8.2−8.3 in the S/E/β2 complex, whereas no further perturbation is observed for [NO[subscript 2]Y[subscript 731]]-α2. Mutations in pathway residues adjacent to the NO[subscript 2]Y that disrupt H-bonding minimally perturb its pK[subscript a]. The pK[subscript a] of NO[subscript 2]Y[subscript 122-]β2 alone or with α2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO[subscript 2]Y]-α2 mutants (2.1−3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pK[subscript a] of the previously reported NO[subscript 2]Y[subscript 356-]β2 (7.5 in the α2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541−14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway. |
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| id | mit-1721.1/72363 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:36:07Z |
| publishDate | 2012 |
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| spelling | mit-1721.1/723632022-09-23T13:10:36Z Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase Yokoyama, Kenichi Uhlin, Ulla Stubbe, JoAnne Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Stubbe, JoAnne Yokoyama, Kenichi Hu, JoAnne E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y[subscript 122-]β2 reversibly oxidizes C[subscript 439] in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y[subscript 122] and Y[subscript 356] in β2, Y[subscript 731] and Y[subscript 730] in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO[subscript 2]Y, pK[subscript a] 7.1) has been incorporated in place of Y[subscript 122], Y[subscript 731], and Y[subscript 730] to probe how the protein environment perturbs each pK[subscript a] in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 × 10[subscript −3] that of the wild-type (wt) subunit. The [NO[subscript 2]Y[subscript 730]]-α2 and [NO[subscript 2]Y[subscript 731]]-α2 each exhibit a pK[subscript a] of 7.8−8.0 with E and E/β2. The pK[subscript a] of [NO[subscript 2]Y[subscript 730]]-α2 is elevated to 8.2−8.3 in the S/E/β2 complex, whereas no further perturbation is observed for [NO[subscript 2]Y[subscript 731]]-α2. Mutations in pathway residues adjacent to the NO[subscript 2]Y that disrupt H-bonding minimally perturb its pK[subscript a]. The pK[subscript a] of NO[subscript 2]Y[subscript 122-]β2 alone or with α2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO[subscript 2]Y]-α2 mutants (2.1−3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pK[subscript a] of the previously reported NO[subscript 2]Y[subscript 356-]β2 (7.5 in the α2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541−14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway. National Institutes of Health (U.S.) (GM29595) 2012-08-28T14:02:23Z 2012-08-28T14:02:23Z 2010-06 2010-02 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/72363 Yokoyama, Kenichi, Ulla Uhlin, and JoAnne Stubbe. “Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a]Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase.” Journal of the American Chemical Society 132.24 (2010): 8385–8397. en_US http://dx.doi.org/10.1021/ja101097p Journal of the American Chemical Society 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 (ACS) PMC |
| spellingShingle | Yokoyama, Kenichi Uhlin, Ulla Stubbe, JoAnne Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase |
| title | Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase |
| title_full | Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase |
| title_fullStr | Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase |
| title_full_unstemmed | Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase |
| title_short | Site-Specific Incorporation of 3-Nitrotyrosine as a Probe of pK[subscript a] Perturbation of Redox-Active Tyrosines in Ribonucleotide Reductase |
| title_sort | site specific incorporation of 3 nitrotyrosine as a probe of pk subscript a perturbation of redox active tyrosines in ribonucleotide reductase |
| url | http://hdl.handle.net/1721.1/72363 |
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