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The heme-undecapeptide microperoxidase-11 (MP-11) was immobilized on mesoporous antimony-doped tin oxide (ATO) thin-film electrodes modified with the positively charged binding promotor polydiallyldimethylammonium chloride. Surface concentrations of MP-11 of 1.5 nmol cm(-2) were sufficiently high to enable spectroelectrochemical analyses. UV/Vis spectroscopy and resonance Raman spectroscopy revealed that immobilized MP-11 adopts a six-coordinated low-spin conformation, as in solution in the presence of a polycation. Cathodic reduction of hydrogen peroxide at potentials close to +500mV versus Ag/AgCl indicates that the reaction proceeds via a Compound I-type like intermediate, analogous to natural peroxidases, and confirms mesoporous ATO as a suitable host material for adsorbing the heme-peptide in its native state. A hydrogen peroxide sensor is proposed by using the bioelectrocatalytic properties of the MP-11-modified ATO.
The homodinuclear ruthenium(II) complex [{Ru(l-N4Me2)}(2)(-tape)](PF6)(4) {[1](PF6)(4)} (l-N4Me2=N,N-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying * orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4Me2)}(2)(-tape(.-))](PF6)(3) {[2](PF6)(3)} and [{Ru(l-N4Me2)}(2)(-tape(2-))](PF6)(2) {[3](PF6)(2)}, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6)(3) contains the -radical tape(.-) and the doubly reduced [3](PF6)(2) the diamagnetic dianion tape(2-) as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1](4+) by two sulfite units gave the complex [{Ru(l-N4Me2)}(2){-tape-(SO3)(2)}](2+) ([4](2+)). Complex dication [4](2+) was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy *-orbital of the disulfonato-substituted bridging ligand tape-(SO3)(2)(2-) (E-red1=-0.1V versus Ag/AgCl/1m KCl in water).
Surface-Tuned Electron Transfer and Electrocatalysis of Hexameric Tyrosine-Coordinated Heme Protein
(2015)
Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant k(s) values between 0.93 and 2.86 s(-1) and apparent formal potentials E-app(0)' between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.
The aim of this thesis is the design, expression and purification of human cytochrome c mutants and their characterization with regard to electrochemical and structural properties as well as with respect to the reaction with the superoxide radical and the selected proteins sulfite oxidase from human and fungi bilirubin oxidase. All three interaction partners are studied here for the first time with human cyt c and with mutant forms of cyt c. A further aim is the incorporation of the different cyt c forms in two bioelectronic systems: an electrochemical superoxide biosensor with an enhanced sensitivity and a protein multilayer assembly with and without bilirubin oxidase on electrodes. The first part of the thesis is dedicated to the design, expression and characterization of the mutants. A focus is here the electrochemical characterization of the protein in solution and immobilized on electrodes. Further the reaction of these mutants with superoxide was investigated and the possible reaction mechanisms are discussed. In the second part of the work an amperometric superoxide biosensor with selected human cytochrome c mutants was constructed and the performance of the sensor electrodes was studied. The human wild-type and four of the five mutant electrodes could be applied successfully for the detection of the superoxide radical. In the third part of the thesis the reaction of horse heart cyt c, the human wild-type and seven human cyt c mutants with the two proteins sulfite oxidase and bilirubin oxidase was studied electrochemically and the influence of the mutations on the electron transfer reactions was discussed. Finally protein multilayer electrodes with different cyt form including the mutant forms G77K and N70K which exhibit different reaction rates towards BOD were investigated and BOD together with the wild-type and engineered cyt c was embedded in the multilayer assembly. The relevant electron transfer steps and the kinetic behavior of the multilayer electrodes are investigated since the functionality of electroactive multilayer assemblies with incorporated redox proteins is often limited by the electron transfer abilities of the proteins within the multilayer. The formation via the layer-by-layer technique and the kinetic behavior of the mono and bi-protein multilayer system are studied by SPR and cyclic voltammetry. In conclusion this thesis shows that protein engineering is a helpful instrument to study protein reactions as well as electron transfer mechanisms of complex bioelectronic systems (such as bi-protein multilayers). Furthermore, the possibility to design tailored recognition elements for the construction of biosensors with an improved performance is demonstrated.