Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy

Abstract

Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.

Ecology and Biotechnology of Selenium-Respiring Bacteria

“…The membrane-bound quinol cytochrome c oxidoreductase (QCR) supplies electrons for SeO 4 2Ϫ reduction. The electrons are shunted to periplasmic cytochrome c 4 (cytc4) and then to SerABC, located in the periplasmic compartment (77,81,149). The reduction of SeO 4 2Ϫ to SeO 3 2Ϫ occurs in the periplasmic compartment.…”

Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis

“…Because SerABC appears to be soluble and not associated with the inner or outer membrane (6), it is likely that another protein acts as a shuttle to take electrons from the membrane and deliver them to the b-heme of SerC. Reduced SerC then donates electrons, via the iron-sulfur clusters of SerAB (11), specifically to the molybdopterin cofactor for reduction of selenate (SeO 4 2Ϫ ) to selenite (SeO 3 2Ϫ ) (6,11,14). The b-heme of SerC is presumed to be the site of electron entry to the selenate reductase (SER) enzyme complex but also remains poorly studied.…”

“…These soluble enzymes consist of three subunits and in addition to the b-heme cytochrome (␥-subunit), they comprise an ironsulfur protein (␤-subunit) coordinating 1 ϫ [3Fe-4S] cluster and 3 ϫ [4Fe-4S] clusters, and a catalytic component (␣-subunit) that coordinates a [4Fe-4S] cluster and the active site molybdopterin guanine dinucleotide cofactor (10,11) (Fig. 1).…”

“…Periplasms were analyzed by SDS-PAGE, and c-type cytochromes were visualized using the heme stain method. SER was purified as detailed by Schröder et al (6) with the modifications noted by Dridge et al (11). Purified enzyme was stored at Ϫ80°C.…”

A bacterial process for selenium nanosphere assembly

“…The selenate reductase (SerABC) isolated from T. selenatis (6) is a soluble periplasmic enzyme. The enzyme is a type II molybdoenzyme that comprises three subunits, SerA (96 kDa), SerB (40 kDa), and SerC (23 kDa), and coordinates molybdenum, heme (b-type), and numerous [Fe-S] centers as prosthetic constituents (9). SerABC contributes to proton-motive force generation by accepting electrons from a diheme c-type cytochrome (cytc 4 ), which mediates electron flux from either a quinol-cytochrome c oxidoreductase (QCR) or quinol dehydrogenase.…”