ELECTRON-TRANSFER AND SPECTRAL ALPHA-BAND PROPERTIES OF THE DI-HEME PROTEIN CYTOCHROME C(4) FROM PSEUDOMONAS-STUTZERI

Cytochrome c(4) is a 190-residue protein active in the aerobic and ana erobic respiration of several bacteria. We have isolated Pseudomonas s tutzeri (ATCC no. 11607) cytochrome c(4) by an optimized growth proced ure following factorial design. The ultraviolet/visible spectra of red uced cytochrome c(4) have a composite alpha/beta band which can be res olved into six components. One of these seems to be specific for the h igh-potential heme group. The kinetics for full oxidation-and reductio n with the two inorganic redox couples, [Co(terpy)(2)](2+/3+) and [Co( bipy)(3)](2+/3+), is formally compatible with either bi- or tri-expone ntial kinetics. The former would be in line with weak interaction betw een the heme groups, the latter with notable interaction effects. Argu ments in favour of the latter and a cooperative two-electron transfer pattern are given. All phases are approximately proportional to the Co -complex concentration, implying that intramolecular electron transfer in this time range is unlikely. The rate constants are in the range ( 0.7-80)x10(4) M(-1) s(-1) at pH = 7.6 (Tris) and 0.1 M NaCl and very l ittle dependent on the ionic strength in the range 0.1-0.3 M. The redu ction potentials could be calculated from the forward and reverse rate constant ratios. The values are 241+/-5 and 328+/-2 mV (Nernst hydrog en electrode) if bi-exponential kinetics is used and interaction betwe en the heme groups disregarded. The intrinsic microscopic reduction po tential values are closer when the tri-exponential, cooperative model is used as this model transfers 30-40 mV to electrostatically dominate d interaction potentials. The overall electron transfer pattern can be related to the recently determined crystal structure of the P. stutze ri cytochrome c(4).