Recent studies in this laboratory on two forms of cytochrome
P450 purified to homogeneity from rabbit liver microsomes are
reviewed. The two forms, phenobarbital-inducible P450LM2 and
5,6-benzoflavone-inducible P450LM4, differ in subunit molecular
weight, identity of the C-terminal amino acid, optical and EPR
spectra, and other properties. As isolated, oxidized P450LM2 is in
the low spin state, whereas P450LM4 is largely, but non entirely, in
the high spin state. Mechanistic studies have shown the following:
(a) P450LM2 may accept two electrons, calculated per heme, from
dithionite or NADPH in the presence of catalytic amounts of the
reductase, and may donate two electrons to various oxidizing agents,
including molecular oxygen. (b) Hydrogen peroxide is formed in
the reconstituted system in the presence of NADPH and oxygen, and
the amount varies with the substrate added. (c) Hydrogen peroxide
and other hydroperoxides apparently donate the oxygen atom
inserted into substrate during hydroxylation in the absence of 0 2
and an external donor. (d) Stopped flow spectrophotometry has
provided evidence for two distinct oxygenated complexes of the
reduced cytochrome. The reductase and cytochrome b5 may play
an effector role in increasing the rate of decomposition of the
second complex during oxygen insertion into substrate. A scheme
is proposed for the mechanism of action of purified P450LM2, based
on these and other findings
