Structural and Biochemical Characterization of a Ferredoxin:Thioredoxin Reductase-like Enzyme from Methanosarcina acetivorans

Abstract

Bioinformatics analyses predict the distribution in nature of several classes of diverse disulfide reductases that evolved from an ancestral plant-type ferredoxin:thioredoxin reductase (FTR) catalytic subunit to meet a variety of ecological needs. Methanosarcina acetivorans is a methane-producing species from the domain Archaea predicted to encode an FTR-like enzyme with two domains, one resembling the FTR catalytic subunit and the other containing a rubredoxin-like domain replacing the variable subunit of present-day FTR enzymes. M. acetivorans is of special interest as it was recently proposed to have evolved at the time of the end-Permian extinction and to be largely responsible for the most severe biotic crisis in the fossil record by converting acetate to methane. The crystal structure and biochemical characteristics were determined for the FTR-like enzyme from M. acetivorans, here named FDR (ferredoxin disulfide reductase). The results support a role for the rubredoxin-like center of FDR in transfer of electrons from ferredoxin to the active-site [Fe₄S₄] cluster adjacent to a pair of redox-active cysteines participating in reduction of disulfide substrates. A mechanism is proposed for disulfide reduction similar to one of two mechanisms previously proposed for the plant-type FTR. Overall, the results advance the biochemical and evolutionary understanding of the FTR-like family of enzymes and the conversion of acetate to methane that is an essential link in the global carbon cycle and presently accounts for most of this greenhouse gas that is biologically generated.

Toward a mechanistic and physiological understanding of a ferredoxin:disulfide reductase from the domains Archaea and Bacteria

“…However, plant ferredoxin:thioredoxin reductase (FTR) is devoid of flavin and contains a novel active-site [Fe 4 S 4 ] cluster (3,4). Methanosarcina acetivorans, classified in the domain Archaea, produces a planttype ferredoxin:disulfide reductase (FDR) also devoid of flavin that contains an active-site [Fe 4 S 4 ] cluster revealed by the crystal structure (5). FDR is representative of a diverse family of disulfide reductases proposed to have evolved from an ancestral plant-type FTR catalytic subunit to meet a variety of specific ecological needs (6,37).…”

“…Preliminary characterization of FDR involved ferredoxin (Fdx)-dependent reduction of GSSG, a nonphysiological substrate absent in methane-producing species from the domain Archaea (methanoarchaea) (5). Although FDR homologs are widely distributed among diverse methanoarchaea, the physiological redox substrates are unknown (6,7).…”

“…1) reveals similarities and differences that necessitate a more comprehensive biochemical investigation of FDR. Both are devoid of flavin and contain a [4Fe-4S] cluster adjacent to the active-site disulfides in FDR (Cys 52 -S-S-Cys 84 ) and FTR (Cys 57 -S-S-Cys 87 ) (3,5). FTR is a ␣␤-heterodimer comprising a variable subunit and a catalytic subunit.…”

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

“…However, it has also been investigated significantly in the anaerobic organisms (6 -24) focusing on both the characteristics of the Trxs and TrxRs (6,8,9,(11)(12)(13)(15)(16)(17)(18)(19)(20)(21)(22)(23) and their physiological roles (6,7,10,14,20). For example, the isolation of the putative Trx targets via Trxaffinity chromatography and the results from activation assays with select purified and deactivated enzymes have established that in Chlorobaculum tepidum, a green sulfur bacterium and an anaerobic phototroph, the thioredoxin system implements post-translational control on the tricarboxylic acid or the TCA cycle and sulfur metabolism and assists in the defense against oxidative stress (7).…”

“…Work with Clostridium pasteurianum, an anaerobic fermentative bacterium, has revealed a ferredoxin-dependent flavin containing TrxR (8). Similarly, there have been several reports on the Trx and TrxRs of anaerobic archaea (9,15,(17)(18)(19)(20)(21)(22)(23), including the methanogenic archaea, which are strict anaerobes (9,17,18,(21)(22)(23). Recently, a proteomics study has shown that there is clear potential for a Trx-based global redox regulation of metabolism, including methanogenesis, in Methanocaldococcus jannaschii, a strictly anaerobic and deeply rooted hyperthermophilic methanogenic archaeon that lives in deep-sea hydrothermal vents (20).…”