Kinetics of Electron Transfer Between Two Hansenula Anomala Flavocytochrome B2 Derivatives and Two Simple Copper Proteins (azurin and Stellacyanin)

Two derivatives of Hansenula anomala flavocytochrome b2 have been prepared, one deprived of the flavin prosthetic group (deflavocytochrome b2), and the other consisting of the heme-b-carrying globule (b2 core). The redox potential of the heme in the two derivatives is -5 (+/- 5) mV and -10 (+/- 5) mV respectively, fairly similar to the value of -20 (+/- 5) mV reported for the holoenzyme, indicating a minor effect of the flavin and of the flavodehydrogenase domain on heme potential. The kinetics of azurin and stellacyanin reduction by both derivatives have been investigated. At pH 7.0, I = 0.2 M and 20 degrees C the second-order rate constants are: k = 8 X 10(5) M-1 S-1 for azurin reduction by deflavocytochrome b2; k = 1.6 X 10(6) M-1 S-1 for azurin reduction by b2 core; k = 1 X 10(7) M-1 S-1 for stellacyanin reduction by deflavocytochrome b2; k = 3 X 10(7) M-1 S-1 for stellacyanin reduction by b2 core. The change in pH markedly affects the kinetics in the case of azurin, but has no effect on stellacyanin reduction. The change in ionic strength has a significant effect when deflavocytochrome b2 is the reductant, indicating that the flavodehydrogenase domain plays a role in the stabilization of the transient kinetic complex by means of electrostatic interactions. The kinetic results are discussed in the framework of the Marcus theory.