Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach.

Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(-1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.

Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer.

An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V (vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 microM sulfite with a sensitivity of 2.19 mA M(-1) sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.

Protein electrodes with direct electrochemical communication.

Electrochemistry using direct electron transfer between an electrode and a protein or an enzyme has developed into a means for studying biological redox reactions and for bioanalytics, biosynthesis and bioenergetics. This review summarizes recent work on direct protein electrochemistry with special emphasis on our results in bioelectrocatalysis using isolated enzymes and enzyme-protein couples.

Electrocatalytically functional multilayer assembly of sulfite oxidase and cytochrome c.

An electrocatalytically functional multilayer has been designed using two proteins, cytochrome c and sulfite oxidase, and a polyelectrolyte (polyaniline sulfonate). The two proteins were co-immobilized on the surface of a gold electrode in alternating layers by electrostatic interactions using the layer-by-layer technique. The formation of this fully electro-active multilayer is characterized by quartz crystal microbalance and electrochemical experiments. The electro-catalytic characterization of the device containing up to 12 layers is based on generation of an oxidation current after sulfite addition. Besides the electron-transfer mechanism, the role of the different components in the electron-transport chain is clarified. Kinetic data were extracted to characterize the multilayer function. This artificial multilayer assembly is expected to be useful in the biosensor and biofuel cell development.