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Abstract
Ferredoxin:thioredoxin reductase (FTR) plays a crucial role in light regulation of oxygenic photosynthesis in chloroplasts. FTR catalyzes the reduction of the disulfide in thioredoxin using a [2Fe-2S] ferredoxin as a one-electron donor, and constitutes a unique class of disulfide reductases that utilizes an active site involving a [4Fe-4S]cluster with an adjacent disulfide. The combination of spectroscopic and mutagenesis studies has been used to investigate the catalytic mechanism of FTR. Wild-type, C57S, C87A, H86Y Synechocystis FTR, as well as a N-ethylmaleimide chemically modified form (NEM-FTR) and a stable heterodisulfide complex formed between FTR and the C40S variant of thioredoxin-m (FTR-Trxm), have been investigated in all accessible redox states using UV-visible absorption, resonance Raman, electron paramagnetic resonance, variable temperature magnetic circular dichroism, and Mssbauer spectroscopies. The results reveal distinct roles for Cys87 and Cys57 that comprise the active-site disulfide. Cys87 functions as an electron transfer thiol that binds to the cluster in the one-electron reduced form to yield a five-coordinate iron site. Cys57 functions as an interchange thiol that forms a heterodisulfide with the thioredoxin substrate. A role for His86 as a proton donor/acceptor is implicated by dramatic changes in the activity and redox properties of the [4Fe-4S] center in the H86Y variant. Mssbauer spectroscopy has been particularly effective in establishing novel site-specific [4Fe-4S] cluster chemistry in the oxidized, one-electron-reduced and two-electron-reduced forms of FTR. The results are consistent with two distinct mechanistic proposals that differ in terms of whether the heterodisulfide intermediate is formed at the one- or two-electron-reduced level. A novel one-3+electron reduced intermediate involving a [4Fe-4S] cluster with a five-coordinate iron site is common to both mechanistic proposals and provides a unique method for cleaving biological disulfides in two sequential one-electron steps.