Role of the [Fe 4 S 4 ] Cluster in Mediating Disulfide Reduction in Spinach Ferredoxin:Thioredoxin Reductase ² Christopher R. Staples, ‡ Eric Gaymard, § Anne-Lise Stritt-Etter, § Joshua Telser, | Brian M. Hoffman, ⊥ Peter Schu ¨rmann, § David B. Knaff, # and Michael K. Johnson* ,‡ Department of Chemistry and the Center for Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602, Laboratoire de Biochimie Ve ´ ge ´ tale, UniVersite ´ de Neucha ˆ tel, CH-2007 Neucha ˆ tel, Switzerland, Chemistry Program, RooseVelt UniVersity, Chicago, Illinois 60605, Department of Chemistry, Northwestern UniVersity, EVanston, Illinois 60208, and Department of Chemistry and Biochemistry, Texas Tech UniVersity, Lubbock, Texas 79409 ReceiVed December 3, 1997 ABSTRACT: Thioredoxin reduction in plant chloroplasts is catalyzed by a unique class of disulfide reductases which use a one-electron donor, [Fe 2 S 2 ] 2+,+ ferredoxin, and has an active site involving a disulfide in close proximity to a [Fe 4 S 4 ] 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., Schu ¨ rmann, 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 [Fe 4 S 4 ] 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. Chloroplast ferredoxin:thioredoxin reductase (FTR) 1 cata- lyzes a key step in light regulation of several carbon assimilation enzymes in oxygenic photosynthesis (1, 2). Electrons are transmitted from the light-harvesting thylakoid membranes to a 2Fe ferredoxin (Fd) and then utilized to cleave the disulfide in thioredoxin f, which in turn activates or deactivates a number of target enzymes by reduction of regulatory disulfide bridges (3). FTR catalyzes the two- electron-reduction of the active-site disulfide of thioredoxin f in sequential one-electron steps with the 2Fe Fd as the one- electron-donor. The enzyme (previously known as ferral- terin) is an R heterodimer. The variable or R subunit (7- 13 kDa) differs in size (4, 5) and shows little or no immunological cross reactivity among different species (6, 7). In contrast, the catalytic or  subunit (13 kDa) is highly conserved among different species (5, 8, 9) and contains the active-site disulfide in close proximity to a [Fe 4 S 4 ] cluster (6, 8, 10). The role and accessibility of the seven conserved cysteine residues in the catalytic subunit has been addressed by extensive studies of spinach FTR with radiolabeled cysteine alkylating agents (8), and the picture of the active site that emerges from these studies is summarized in Figure 1. The active-site disulfide (Cys54-S-S-Cys84) is clearly in close proximity to the [Fe 4 S 4 ] cluster, since a single residue ² This work was supported by grants from the National Institutes of Health (GM51962 to M.K.J. and HL13531 to B.M.H.), the National Science Foundation (DMB-8907559 to B.M.H.), the Schweizerischer Nationalfonds (31-47107.96 and 31-37725.93 to P.S.), the U.S. Department of Energy (DE-FG03-93ER20125 to D.B.K.), and a NSF Research Training Group Award to the Center for Metalloenzyme Studies (BIR9413236 to M.K.J.). * Author to whom correspondence should be addressed. Tele- phone: (706) 542-9378. Fax: (706) 542-2353. E-mail: johnson@ sunchem.chem.uga.edu. ‡ University of Georgia. § Universite ´ de Neucha ˆtel. | Roosevelt University. ⊥ Northwestern University. # Texas Tech University. 1 Abbreviations: FTR, ferredoxin:thioredoxin reductase; T, thiore- doxin; NEM, N-ethylmaleimide; NEM-FTR, a modified form of FTR with NEM covalently attached at one of the cysteines of the active- site disulfide; (VT)MCD, (variable-temperature) magnetic circular dichroism; RR, resonance Raman; ENDOR, electron nuclear double resonance; Fd, ferredoxin; NHE, normal hydrogen electrode; CW, continuous wave; DTT, dithiothreitol; HiPIP, high potential iron-sulfur protein. 4612 Biochemistry 1998, 37, 4612-4620 S0006-2960(97)02976-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/17/1998