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

Elizabeth J Dridge , Carys A Watts , Brian J Jepson , Kirsty Line , Joanne M Santini , David J Richardson , Clive S Butler (1)

1 School of Biosciences

Selenate reductase (SerABC) from Thauera selenatis is classified as a member of the Tat-translocated (Type II) molybdo-enzymes and comprises three subunits each containing redox cofactors. Variable temperature X-band electron magnetic resonance (EPR) spectra of the purified selenate reductase showed complex features attributable to centres [3Fe-4S] 1+} [4Fe-4S] 1+}, Mo(V) and haem-b. EPR monitored redox-potentiometric titration of SerABC complex (hetero-trimetric complex of αβγ subunits) revealed that the [3Fe-4S] cluster (FS4) titrated as n = 1 Nernstian component with a mid-point 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 mid-point potential of E m}~ -356 mV (FS2). It is considered likely that at low redox potential E m}< -300 mV, the remaining oxidised cluster is reduced (S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S] 1+} cluster rendering both centres silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of selenate reductase 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 g 3}~1.999, g 2}~1.996 and g 1}~1.965 (g av} 1.9867). The dominant features at g 2} and g 3} are not split, but hyperfine splitting is observed in the g 1} region of the spectrum and can be simulated as arising from a single proton with a coupling constant of A 1} ( 1}H) = 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 selenate reductase as a member of the Type II molybdo-enzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.

Mots clés: Life Sciences

Elizabeth J Dridge, Carys A Watts, Brian J Jepson, Kirsty Line, Joanne M Santini, et al.. Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy. Biochemical Journal, 2007, 408 (1), pp.19-28. ⟨10.1042/BJ20070669⟩. ⟨hal-00478807⟩