PEROXO-IRON AND OXENOID-IRON SPECIES AS ALTERNATIVE OXYGENATING AGENTS IN CYTOCHROME P450-CATALYZED REACTIONS - SWITCHING BY THREONINE-302 TO ALANINE MUTAGENESIS OF CYTOCHROME-P450 2B4
Adn. Vaz et al., PEROXO-IRON AND OXENOID-IRON SPECIES AS ALTERNATIVE OXYGENATING AGENTS IN CYTOCHROME P450-CATALYZED REACTIONS - SWITCHING BY THREONINE-302 TO ALANINE MUTAGENESIS OF CYTOCHROME-P450 2B4, Proceedings of the National Academy of Sciences of the United Statesof America, 93(10), 1996, pp. 4644-4648
Among biological catalysts, cytochrome P450 is unmatched in its multip
licity of isoforms, inducers, substrates, and types of chemical reacti
ons catalyzed. In the present study, evidence is given that this versa
tility extends to the nature of the active oxidant. Although mechanist
ic evidence from several laboratories points to a hypervalent iron-oxe
noid species in P450-catalyzed oxygenation reactions. Akhtar and colle
agues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982)
Biochem, J. 201, 569-580] proposed that in steroid deformylation effe
cted by P450 aromatase an iron-peroxo species is involved. We have sho
wn more recently that purified liver microsomal p450 cytochromes, incl
uding phenobarbital-induced p450 2B4, catalyze the analogous deformyla
tion of a series of xenobiotic aldehydes with olefin formation. The in
vestigation presented here on the effect of site-directed mutagenesis
of threonine-302 to alanine on the activities of recombinant P450 2B4
with N-terminal amino acids 2-27 deleted [2B4 (Delta 2-27)] makes use
of evidence from other laboratories that the corresponding mutation in
bacterial P450s interferes with the activation of dioxygen to the oxe
noid species by blocking proton delivery to the active site. The rates
of NADPH oxidation, hydrogen peroxide production, and product formati
on from four substrates, including formaldehyde from benzphetamine N-d
emethylation, acetophenone from 1-phenylethanol oxidation, cyclohexano
l from cyclohexane hydroxylation, and cyclohexene from cyclohexane car
boxaldehyde deformylation, were determined with P450s 2B4, 2B4 (Delta
2-27), and 2B4 (Delta 2-27) T302A. Replacement of the threonine residu
e in the truncated cytochrome gave a 1.6- to 2.5-fold increase in pero
xide formation in the presence of a substrate, but resulted in decreas
ed product formation from benzphetamine (9 fold), cyclohexane (4-fold)
, and 1-phenylethanol (2-fold). In sharp contrast, the deformylation o
f cyclohexane carboxaldehyde by the T302A mutant was increased about 1
0-fold. On the basis of these findings and our previous evidence that
aldehyde deformylation is supported by added H2O2, but not by artifici
al oxidants, we conclude that the iron-peroxy species is the direct ox
ygen donor. It remains to be established which of the many other oxida
tive reactions involving P450 utilize this species and the extent to w
hich peroxo-iron and oxenoid-iron function as alternative oxygenating
agents with the numerous isoforms of this versatile catalyst.