Biocatalysis on Nanostructured Surfaces: Investigation and Application of Redox Proteins using Spectro-Electrochemical Methods

So far, the electrochemistry of sulfite oxidizing enzymes through the direct and mediated communication of heme b5 has been investigated. The electrochemical behavior of SO was also reported using different synthetic mediators and cyt c (Coury Jr. et al., 1991). All employed mediators were able to communicate only via the heme b5. The IET reaction of SO was indicated as the limiting step (10 s-1) of the overall ET (Coury Jr. et al., 1991). Direct electrochemical communication of gallus gallus SO with electrodes has been demonstrated (Elliott et al., 2002; Ferapontova et al., 2003). After immobilization on pyrolytic graphite or gold electrode modified with mercapto-6-hexanol self-assembled monolayer the enzyme can partially retain its activity and a single pair of peaks were observed with a formal potential of + 90 mV (vs. standard hydrogen electrode, SHE). Upon addition of sulfite to the cell solution, the voltammogram changed to an oxidative catalytic wave. However, the immobilized SO showed a decreased catalytic turnover rate (2-4 s-1) in comparison to SO in solution with cyt c (100 s-1) (Elliott et al., 2002). This behavior was attributed to the small fraction of cSO molecules in with an appropriate conformation electrons exchange of the HD with the electrode surface. A faster heterogeneous electron transfer of cSO immobilized was reached on aminated surface mimicking the natural partner of the enzyme (cyt c). Using gold surface modified with 11-mercapto-1-undecanol and 11-mercapto-1-undecanamine the ket was in the order of 15 s-1, whereas the catalytic turnover rate was comparable to the reaction in solution with the natural electron acceptor cyt c (Ferapontova et al., 2003). The E0 was to −120 mV versus Ag/AgCl (KClsat), correlating fairly well with the reported redox potential for the heme Fe(III/II) couple in SO obtained through optical redox titration (Spence et al., 1991; Ferapontova et al., 2004). The E0 for MoVI/V and MoV/IV interpolated at pH 8 were +6 mV and -184 mV (vs. NHE), hence the half reduced state of the active site (MoV) is stable (Spence et al., 1991). These data are in agreement with the IET rates determined at pH 8 using flash photolysis on the same enzyme (Sullivan Jr. et al., 1993).
CSO has been employed for the constitution of first (Situmorang et al., 1999) and second (Abass, 2000; Svitel et al., 1998) generation of sulfite biosensors. Only recently the research is addressing the human variant of the sulfite oxidase enzyme. Three different approaches for the fabrication of a sulfite biosensing system have been proposed. In one case the natural partner of the hSO was employed for the electrochemical mediation with the electrode. Complex protein architectures containing hSO and cyt c were construct by the layer-by-layer technique (Dronov et al., 2008) as well as alternated by an anionic polyelectrolyte (Spricigo et al., 2008) (Spricigo et al., 2009). It resulted in an enhanced sensitivity towards the sulfite concentration. The system showed appreciable stability only at very low ionic strength.