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 αβγ subunits) revealed that the [3Fe–4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (Em) 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 (Em) of about −356 mV (FS2). It is considered likely that, at low redox potential (Em 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∼1.999, g2∼1.996 and g1∼1.965 (gav 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∼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.

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