Kinetics and mechanism of electron transfer from dithionite to microsomal cytochrome b5 and to forms of the protein associated with charged and neutral vesicles.
The kinetics of the dithionite reduction of calf liver microsomal cytochrome b5, both free in solution and bound to dimyristoyl phosphatidylcholine vesicles, are consistent with electron transfer between SO2- and the exposed haem edge of the protein. The vesicle membrane does not hinder the approach of SO2- to the site of electron transfer on the protein. In 0.01 M-Tris/HCl buffer, pH 8.1, ket (25 degrees C), delta H et and delta S et are estimated to be 1.44 x 10(6) M-1.s-1, 7.8 kJ.mol-1 and -92.3 J.K-1.mol-1 respectively. The cytochrome exhibits an acid dissociation, pKa 9.3 +/- 0.3, and the rate of electron transfer from dithionite to the high-pH form is about one-third of that to the neutral-pH form. The effect of ionic strength on the kinetics is consistent with a reaction between like-charged species and is discussed in terms of a number of theoretical models. In systems comprising cytochrome b5 and negatively charged vesicles, the effect of increasing the charge density of mixed dimyristoyl phosphatidylcholine/dicetyl phosphate vesicles and of increasing the concentration of dicetyl phosphate vesicles is to lower the rate of electron transfer from dithionite to the haem moiety of the cytochrome. With vesicles of high charge density, however, the kinetics are complicated by vesicle-induced conformation changes of the cytochrome.
Davies
D M
DM
Department of Chemical and Life Sciences, Newcastle upon Tyne Polytechnic, U.K.
Lawther
J M
JM
D000818:Animals; D002417:Cattle; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004227:Dithionite / Q000378:metabolism*; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008566:Membranes; D008862:Microsomes, Liver / Q000378:metabolism*; D009994:Osmolar Concentration; D012964:Sodium / Q000378:metabolism; D013447:Sulfites / Q000378:metabolism*; D013696:Temperature
D003573:Cytochrome b Group; D013447:Sulfites; D004227:Dithionite; D015786:Cytochromes b5; D012964:Sodium; D004134:Dimyristoylphosphatidylcholine