Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase
Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse t...
| Main Authors: | , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
Royal Society of Chemistry
2016
|
| Online Access: | http://hdl.handle.net/1721.1/103948 https://orcid.org/0000-0001-8076-4489 |
| _version_ | 1826197694824054784 |
|---|---|
| author | Song, David Y. Pizano, Arturo A. Holder, Patrick G. Stubbe, JoAnne Nocera, Daniel G. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Song, David Y. Pizano, Arturo A. Holder, Patrick G. Stubbe, JoAnne Nocera, Daniel G. |
| author_sort | Song, David Y. |
| collection | MIT |
| description | Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β[subscript 2] and α[subscript 2]) involving multiple PCET steps and the conserved tyrosine triad [Y[subscript 356](β[subscript 2])–Y[subscript 731](α[subscript 2])–Y[subscript 730](α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y[subscript 356]C-(β[subscript 2]) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F[subscript 3]Y) is incorporated in place of Y[subscript 731] in α[subscript 2]. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re[subscript 35]6]-β[subscript 2]:2,3,5-F[subscript 3]Y[subscript 731]-α[subscript 2] in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α[subscript 2] from β[subscript 2] in the absence of the interfacial Y[subscript 356] residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y[subscript 731](α[subscript 2]), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y[subscript 356] and Y[subscript 731], at the subunit interface. |
| first_indexed | 2024-09-23T10:51:43Z |
| format | Article |
| id | mit-1721.1/103948 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:51:43Z |
| publishDate | 2016 |
| publisher | Royal Society of Chemistry |
| record_format | dspace |
| spelling | mit-1721.1/1039482022-09-27T15:32:39Z Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase Direct interfacial Y731 oxidation in α2 by a photoβ2 subunit of E. coli class Ia ribonucleotide reductase Song, David Y. Pizano, Arturo A. Holder, Patrick G. Stubbe, JoAnne Nocera, Daniel G. Massachusetts Institute of Technology. Department of Chemistry Stubbe, JoAnne Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β[subscript 2] and α[subscript 2]) involving multiple PCET steps and the conserved tyrosine triad [Y[subscript 356](β[subscript 2])–Y[subscript 731](α[subscript 2])–Y[subscript 730](α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y[subscript 356]C-(β[subscript 2]) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F[subscript 3]Y) is incorporated in place of Y[subscript 731] in α[subscript 2]. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re[subscript 35]6]-β[subscript 2]:2,3,5-F[subscript 3]Y[subscript 731]-α[subscript 2] in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α[subscript 2] from β[subscript 2] in the absence of the interfacial Y[subscript 356] residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y[subscript 731](α[subscript 2]), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y[subscript 356] and Y[subscript 731], at the subunit interface. National Science Foundation (U.S.) (Division of Graduate Education Grant DGE-1144152) National Science Foundation (U.S.) (Post-Doctoral Fellowship (GM087034)) National Institutes of Health (U.S.) (grant GM047274) National Institutes of Health (U.S.) (NIH grant GM029595) 2016-08-17T18:41:48Z 2016-08-17T18:41:48Z 2015-06 2015-03 Article http://purl.org/eprint/type/JournalArticle 2041-6520 2041-6539 http://hdl.handle.net/1721.1/103948 Song, David Y., Arturo A. Pizano, Patrick G. Holder, JoAnne Stubbe, and Daniel G. Nocera. "Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase." Chemical Science 6 (2015), pp. 4519-4524. https://orcid.org/0000-0001-8076-4489 en_US http://dx.doi.org/10.1039/c5sc01125f Chemical Science Creative Commons Attribution 3.0 Unported licence http://creativecommons.org/licenses/by/3.0/ application/pdf Royal Society of Chemistry Royal Society of Chemistry |
| spellingShingle | Song, David Y. Pizano, Arturo A. Holder, Patrick G. Stubbe, JoAnne Nocera, Daniel G. Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase |
| title | Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase |
| title_full | Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase |
| title_fullStr | Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase |
| title_full_unstemmed | Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase |
| title_short | Direct interfacial Y[subscript 731] oxidation in α[subscript 2] by a photoβ[subscript 2] subunit of E. coli class Ia ribonucleotide reductase |
| title_sort | direct interfacial y subscript 731 oxidation in α subscript 2 by a photoβ subscript 2 subunit of e coli class ia ribonucleotide reductase |
| url | http://hdl.handle.net/1721.1/103948 https://orcid.org/0000-0001-8076-4489 |
| work_keys_str_mv | AT songdavidy directinterfacialysubscript731oxidationinasubscript2byaphotobsubscript2subunitofecoliclassiaribonucleotidereductase AT pizanoarturoa directinterfacialysubscript731oxidationinasubscript2byaphotobsubscript2subunitofecoliclassiaribonucleotidereductase AT holderpatrickg directinterfacialysubscript731oxidationinasubscript2byaphotobsubscript2subunitofecoliclassiaribonucleotidereductase AT stubbejoanne directinterfacialysubscript731oxidationinasubscript2byaphotobsubscript2subunitofecoliclassiaribonucleotidereductase AT noceradanielg directinterfacialysubscript731oxidationinasubscript2byaphotobsubscript2subunitofecoliclassiaribonucleotidereductase AT songdavidy directinterfacialy731oxidationina2byaphotob2subunitofecoliclassiaribonucleotidereductase AT pizanoarturoa directinterfacialy731oxidationina2byaphotob2subunitofecoliclassiaribonucleotidereductase AT holderpatrickg directinterfacialy731oxidationina2byaphotob2subunitofecoliclassiaribonucleotidereductase AT stubbejoanne directinterfacialy731oxidationina2byaphotob2subunitofecoliclassiaribonucleotidereductase AT noceradanielg directinterfacialy731oxidationina2byaphotob2subunitofecoliclassiaribonucleotidereductase |