Role of the [Fe4S4] cluster in mediating disulfide reduction in spinach ferredoxin:thioredoxin reductase

Christopher R. Staples, Eric Gaymard, Anne Lise Stritt-Etter, Joshua Telser, Brian M. Hoffman, Peter Schürmann, David B. Knaff, Michael K. Johnson*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

Thioredoxin reduction in plant chloroplasts is catalyzed by a unique class of disulfide reductases which use a one-electron donor, [Fe2S2]2+.+ ferredoxin, and has an active site involving a disulfide in close proximity to a [Fe4S4]2+ cluster. In this study, spinach ferredoxin:thioredoxin reductase (FTR) reduced with stoichiometric amounts of reduced benzyl viologen or frozen under turnover conditions in the presence of thioredoxin is shown to exhibit a slowly relaxing S = 1/2 resonance (g = 2.11, 2.00, 1.98) identical to that of a modified form of the enzyme in which one of the cysteines of the active-site disulfide is alkylated with N- ethylmaleimide (NEM-FTR). Hence, in accord with the previous proposal [Staples, C.R., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A.-L., Schurmann, P., Knaff, D.B., and Johnson, M.K. (1996) Biochemistry 35, 11425- 11434], NEM-FTR is shown to be a stable analogue of a one-electron-reduced enzymatic intermediate. The properties of the Fe-S cluster in NEM-FTR have been further investigated by resonance Raman and electron nuclear double resonance spectroscopies; the results, taken together with the previous UV- visible absorption, variable temperature magnetic circular dichroism, and resonance Raman data, indicate the presence of a novel type of [Fe4S4]3+ cluster that is coordinated by five cysteinates with little unpaired spin density delocalized onto the cluster-associated cysteine of the active-site disulfide. While the ligation site of the fifth cysteine remains undefined, the best candidate is a cluster bridging sulfide. On the basis of the spectroscopic and redox results, mechanistic schemes are proposed for the benzyl viologen-mediated two-electron-reduction of FTR and the catalytic mechanism of FTR. The catalytic mechanism involves novel S-based cluster chemistry to facilitate electron transfer to the active-site disulfide resulting in covalent attachment of the electron-transfer cysteine and generation of the free interchange cysteine that is required for the thiol- disulfide interchange reaction with thioredoxin.

Original languageEnglish (US)
Pages (from-to)4612-4620
Number of pages9
JournalBiochemistry
Volume37
Issue number13
DOIs
StatePublished - Mar 31 1998

ASJC Scopus subject areas

  • Biochemistry

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