Resolution of the four hemes of cytochrome c554 from Nitrosomonas europaea by redox potentiometry and optical spectroscopy.

The electrochemical behavior of tetraheme cytochrome c554 from Nitrosomonas europaea has been studied by thin-layer spectroelectrochemistry, cyclic voltammetry, differential pulse voltammetry, and alternating current voltammetry. Three redox couples were detected. Midpoint potentials for the high-m intermediate-, and low-potential couples are +47, -147, and -276 mV, respectively, from the spectroelectrochemical measurements and +50, -120, and -225 mV, respectively, from the voltammetry measurements. A coulometric titration shows that two electrons are taken up by the high-potential couple and one each is taken up by the intermediate- and low-potential couples. Results from the spectroelectrochemical titration at carefully chosen wavelengths indicate that the intermediate- and low-potential couples obey simple Nernstian behavior. The electrochemical behavior of the high-potential couple is apparently not truly Nernstian but is most consistent with two sites exhibiting slight positive cooperativity. Spectral changes associated with the three couples reveal distinctive features in the reduced-minus-oxidized difference spectra. The difference spectrum of the high-potential pair of hemes suggest a mixture of a high-spin heme and a low-spin heme with maxima at 424 and 432 nm. The difference spectrum of the intermediate-potential heme is low spin with a split Soret with maxima at 414 and 424 nm. The difference spectrum of the low-potential heme also shows a split Soret with maxima at 418 and 432 nm.