Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase
Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique proper...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
| Language: | English |
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American Chemical Society
2018
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| _version_ | 1797095050977476608 |
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| author | Evans, R Ash, P Beaton, S Brooke, E Vincent, K Carr, S Armstrong, F |
| author_facet | Evans, R Ash, P Beaton, S Brooke, E Vincent, K Carr, S Armstrong, F |
| author_sort | Evans, R |
| collection | OXFORD |
| description | Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe–3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states “Nia-R” and “Nia-C”, even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, “Ni-B”, is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes. |
| first_indexed | 2024-03-07T04:22:30Z |
| format | Journal article |
| id | oxford-uuid:cb7e672c-f32f-4986-9e62-43df808d19c8 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:22:30Z |
| publishDate | 2018 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | oxford-uuid:cb7e672c-f32f-4986-9e62-43df808d19c82022-03-27T07:15:15ZMechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenaseJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:cb7e672c-f32f-4986-9e62-43df808d19c8EnglishSymplectic Elements at OxfordAmerican Chemical Society2018Evans, RAsh, PBeaton, SBrooke, EVincent, KCarr, SArmstrong, FCatalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe–3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states “Nia-R” and “Nia-C”, even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, “Ni-B”, is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes. |
| spellingShingle | Evans, R Ash, P Beaton, S Brooke, E Vincent, K Carr, S Armstrong, F Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase |
| title | Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase |
| title_full | Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase |
| title_fullStr | Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase |
| title_full_unstemmed | Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase |
| title_short | Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O2-tolerant [NiFe]-hydrogenase |
| title_sort | mechanistic exploitation of a self repairing blocked proton transfer pathway in an o2 tolerant nife hydrogenase |
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