ELECTROCHEMISTRY OF CYTOCHROME C(4) FROM PSEUDOMONAS-STUTZERI

The cyclic voltammetry of the dipolar, overall negatively charged bact erial di-heme protein cytochrome c(4) from Pseudomonas stutzeri is com posite and shows nontraditional features. Close to reversible voltamme try, with individual peaks corresponding to electron exchange of each heme, is found at high ionic strength (0.1 M phosphate, pH = 7.5) usin g gold electrodes modified by any of the promoters 4,4'-bipyridyl disu lfide (4,4'-bpySS), 2,2'-dithiobisethaneamine (cystamine), and 3-pyrid inylmethylenehydrazine carbothiamide. These are otherwise known to pro mote electrochemistry of proteins with positive, negative, and either positive or negative overall charges, respectively. This observation i s indicative of weak surface orientational selectivity of cyt c(4) at high ionic strength. In contrast, the voltammograms at low ionic stren gth (0.01 M phosphate, pH 7.5) point toward orientational selectivity in accord with the expected charge compatibility of the promoters with given domains of the protein. Numerical analysis of the voltammograms provide first macroscopic midpoint potentials and interfacial electro n-transfer (ET) rate constants of each heme. The midpoint potentials a t high buffer concentration are close to values previously determined from ET kinetics in homogeneous solution. At low ionic strength where orientational selectivity at 4,4'-bpySS- and cystamine-modified electr odes is likely, intramolecular ET between the heme groups is, secondly , a feature of the overall interfacial kinetics. The intramolecular ra te constants can be determined from the voltammetric peak shapes, givi ng 40 s(-1) for ET from the high- to the low-potential heme and 1600 s (-1) for the reverse reaction. These values hold interesting implicati ons in relation to the electronic structure and ET patterns in homogen eous solution.