Details

Autor(en) / Beteiligte

• Rapson, Trevor D.
• Kappler, Ulrike
• Hanson, Graeme R.
• Bernhardt, Paul V.

Titel

Short circuiting a sulfite oxidising enzyme with direct electrochemistry: Active site substitutions and their effect on catalysis and electron transfer

Ist Teil von

• Biochimica et biophysica acta, 2011, Vol.1807 (1), p.108-118

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2011

Quelle

MEDLINE

Beschreibungen/Notizen

• Sulfite dehydrogenase (SDH) from Starkeya novella is a heterodimeric enzyme comprising a Mo active site and a heme c electron relay, which mediates electron transfer from the Mo cofactor to cytochrome c following sulfite oxidation. Studies on the wild type enzyme (SDH WT) and its variants have identified key amino acids at the active site, specifically Arg-55 and His-57. We report the Mo VI/V, Mo V/IV and Fe III/II (heme) redox potentials of the variants SDH R55K, SDH R55M, SDH R55Q and SDH H57A in comparison with those of SDH WT. For SDH R55M, SDH R55Q and SDH H57A the heme potentials are lowered from ca. 240 mV in SDH WT to ca. 200 mV, while the heme potential in SDH R55K remains unchanged and the Mo redox potentials are not affected significantly in any of these variants. Protein film voltammetry reveals a pH dependence of the electrochemical catalytic half-wave potential ( E cat ) of −59 mV/pH in SDH WT and SDH R55K which tracks the pH dependence of the Mo VI/V redox potential. By contrast, the catalytic potentials for SDH R55M and SDH H57A are pH-independent and follow the potential of the heme cofactor. These results highlight a switch in the pathway of electron exchange as a function of applied potential that is revealed by protein film voltammetry where an actuation of rate limiting intramolecular electron transfer (IET, Mo to heme) at high potential attenuates the catalytic current relative to faster, direct electron transfer (Mo to electrode) at lower potential. The same change in electron transfer pathway is linked to an unusual peak-shaped profile of the ideally sigmoidal steady state voltammogram in SDH WT alone, which has been associated with a potential dependent change in the orientation of the enzyme on the electrode surface. All other variants show purely sigmoidal voltammetry due to their inherently slower turnover numbers which are always lower than IET rates. [Display omitted] ► Arg-55 and His-57 play a crucial role in SDH catalysis. ► Loss of positive charge in SDH R55M lowers heme potential by 40 mV. ► SDH WT electrochemical catalysis bypasses higher potential heme cofactor. ► Unusual waveforms indicate potential dependent electron transfer pathway.