Expression, purification, and physical properties of recombinant flavocytochrome fusion proteins containing rat cytochrome b(5) linked to NADPH-cytochrome P450 reductase by different membrane-binding segments
Aa. Gilep et al., Expression, purification, and physical properties of recombinant flavocytochrome fusion proteins containing rat cytochrome b(5) linked to NADPH-cytochrome P450 reductase by different membrane-binding segments, ARCH BIOCH, 390(2), 2001, pp. 222-234
Reconstitution of the enzymatic activities using purified microsomal cytoch
rome P450s (P450) requires the presence of a membrane-binding segment in th
e mammalian flavoprotein, NADPH-cytochrome P450 reductase (CPR), and the he
meprotein, cytochrome b(5) (b(5)). The mechanism(s) by which the membrane-b
inding segments of these proteins exert such a critical role in influencing
the reconstitution of the NADPH-supported activity of a P450 remains undef
ined. In the present work we describe the construction, expression, and pur
ification of four different types of recombinant flavocytochromes containin
g rat b(5) and rat CPR linked by various membrane-binding segments, The phy
sical properties of these artificial fusion proteins have been studied to d
etermine their ability to serve as electron transfer agents, These studies
are a prelude to the subsequent study (accompanying paper) evaluating the f
unctional roles of the hydrophobic (membrane-binding) sequences of b(5) and
CPR in the reconstitution of P450 activities. The present study shows that
the purified recombinant fusion proteins can serve as active electron tran
sport carriers from NADPH to cytochrome c as well as b(5) by intramolecular
as well as intermolecular reactions. It is shown here that the electron tr
ansport properties of these purified fusion proteins are influenced by high
concentrations of KCI, suggesting a role for charged amino acids in protei
n-protein interactions. The present study illustrates the application of ar
tificial recombinant flavocytochromes as useful proteins for the study of i
ntramolecular electron transport reactions for comparison with intermolecul
ar interactions. (C) 2001 Academic Press.