Kinetic study on the successive four-step reduction of Cyt c3

A detailed kinetic study on the successive four-step reduction of cyt c3, which has four heme units in a single protein, III4 leads to III3II leads to III2II2 leads to III II3 leads to II4, was carried out by stopped-flow electronic spectroscopy (SF-UV) and stopped-flow circular dichroism spectroscopy (SF-CD). Based on the absorbance change vs. time and the ellipticity change vs. time at the characteristic CD, together with the electronic absorption of the enzyme, rate constants for the successive four electron transfer steps, k1-k4, were successfully estimated by computer simulation. The rate constants of the four steps (k1 = 19.8 s-1, k2 = 11.9 s-1, k3 = 8.9 s-1, and k4 = 1.6 s-1; 8.0 10(-4) M Na2S2O4) are quite different from the statistical values (4: 3: 2: 1), thus excluding the possibility of random reduction of hemes of equal reactivities. Instead, each heme has its own reactivity, probably dependent on its local environment. The value of k3 is somewhat higher than the statistical value, indicating the existence of an autoacceleration effect, although small. This autoacceleration is most probably due to a unique heme-heme and/or heme-environment interaction since unusual CD and electronic absorptions were observed at 350-400 nm at about the time corresponding.