Direct electrochemical studies of cytochromes b(562)

The electrochemistry of cytochrome b(562) from E. coli has been studied at graphite and gold electrodes. At edge plane graphite surfaces neomycin promotes reversible, linear-diffusion limited electrochemistry across the pH range 4.5 to 8.0. At gold surfaces, a similar, but much more stable response is observed when the electrode is electrochemically modified with the cationic hexapeptide, KCTCCA. This reversible direct electrochemical response has been used to study the reduction potential of wild type cytochrome b(562) as a function of pH, ionic strength and temperature. The enthalpic and entropic parameters for the redox equilibrium show a dependence on pH consistent with the idea that the protonation of the haem propionate groups contribute significantly to the control of reduction equilibrium. The electrochemical response from the wild type protein is not reversible above pH 8 and is not detectable above pH 9. This is rationalised by the presence of a species with a deprotonated histidine ligand in the ferricytochrome but surprisingly, we have not observed any electrochemistry attributable to this species under the conditions employed in this work. We have also studied variants of this cytochrome which have bis-methionine ligation and undergo redox Linked ligation state changes between low-spin and high-spin ferric species. The consequences of these changes for the electrochemical response are examined.