Interfacial chemistry of cytochrome C: Substitution of ruthenium complexes and orientation of electrode surfaces

The substitution reaction of racemic ruthenium complexes cis -[Ru(L-L)2(H2O)2] 2+ (L-L = 2,2'-bipyridine (bpy) or 4,4 '-dimethyl-2,2'-bipyridine (dmbpy)) on the surface His 33 residue and the more buried His 26 residues of Horse heart cytochrome c (cyt c) takes place in a stereoselective manner. Whereas large enantiomeric excess (ee) for Delta-[Ru(dmbpy) 2(H2O)]-His26-cyt c (38%), only small ee for Delta-[Ru(bpy)2(H2O)]-His26-cyt c (6%) are observed. Similar ee (∼30%) for both bpy and dmbpy ruthenium complexes modified proteins, Λ-[Ru(L-L)2(H2O)]-His33-cyt c, were observed. Different redox potentials were determined for the different isomers with current ratios consistent with the circular dichroism of the ruthenated proteins. The rates of substitution of imidazole on ruthenium aquo proteins is shown to proceeds with retention of configuration at the ruthenium site. For different ruthenium protein isomers, substitution rates of isomers determined were one to two orders of magnitude slower than those with the corresponding small molecule racemic complexes.;Pulse radiolysis technique provided the method of choice for studying rates of intraprotein electron transfer (ET) in the non-light-sensitive complexes such as [Ru(dabpy)2L]-His33-cyt c series (dabpy = 4,4'-diamino-2,2'-bipyridine, L = N-heterocycle ligands). These newly measured ET rates spanned over two orders of magnitude and were in the linear part of the Marcus parabolic rate vs. free energy plot for this reaction. The stereoselectivity exhibited by the [Ru(L-L)2(H2O)]-His33-cyt c resulted in the formation of multiple isomers which complicated the measurement of the intramolecular ET processes for the aquo complexes of the modified proteins. The ET kinetics of pure [Ru(bpy)2Im]- His33-cyt c studied by pulse radiolysis was found to be consistent with those studied by flash photolysis.;The second part of this thesis is concerned with immobilization and orientation of cyt c on electrode surfaces. In order to achieve this, different methods for preparing a variety of trans-tetraammine-ruthenium complexes on self-assembled monolayer (SAM) surfaces on gold electrodes were investigated. A cyt c monolayer with controlled orientation was assembled by using lipoic acid tethered tetraammine-ruthenium yeast iso-1 cyt c modified at His 39. The electrical interaction of cyt c is mediated by the ruthenium complex, and the protein viability is demonstrated by enzymatic turnover of cyt c oxidase in the presence of oxygen. In this specific design, the redox potential of the ruthenium mediator made the monolayer more amenable to using reduced cyt c in such reactions as those with cyt c oxidase.