Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry.
Rapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters dis...
| Main Authors: | , |
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| Format: | Journal article |
| Language: | English |
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2000
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| _version_ | 1797074972810674176 |
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| author | Camba, R Armstrong, F |
| author_facet | Camba, R Armstrong, F |
| author_sort | Camba, R |
| collection | OXFORD |
| description | Rapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two [4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from [3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal [3Fe-4S] formation (in excess over [4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The [3Fe-4S]:[4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that [3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of [4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with [3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of [3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to [4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the [3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts. |
| first_indexed | 2024-03-06T23:43:54Z |
| format | Journal article |
| id | oxford-uuid:703f1526-678d-49ff-9387-e986828e0359 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T23:43:54Z |
| publishDate | 2000 |
| record_format | dspace |
| spelling | oxford-uuid:703f1526-678d-49ff-9387-e986828e03592022-03-26T19:35:53ZInvestigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:703f1526-678d-49ff-9387-e986828e0359EnglishSymplectic Elements at Oxford2000Camba, RArmstrong, FRapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two [4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from [3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal [3Fe-4S] formation (in excess over [4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The [3Fe-4S]:[4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that [3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of [4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with [3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of [3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to [4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the [3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts. |
| spellingShingle | Camba, R Armstrong, F Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. |
| title | Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. |
| title_full | Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. |
| title_fullStr | Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. |
| title_full_unstemmed | Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. |
| title_short | Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry. |
| title_sort | investigations of the oxidative disassembly of fe s clusters in clostridium pasteurianum 8fe ferredoxin using pulsed protein film voltammetry |
| work_keys_str_mv | AT cambar investigationsoftheoxidativedisassemblyoffesclustersinclostridiumpasteurianum8feferredoxinusingpulsedproteinfilmvoltammetry AT armstrongf investigationsoftheoxidativedisassemblyoffesclustersinclostridiumpasteurianum8feferredoxinusingpulsedproteinfilmvoltammetry |