Electron paramagnetic resonance and temperature dependent spin state studies of ferric cytochrome P-450 from Rhizobium japonicum

Abstract

Cytochrome P-450 as isolated from Rhizobium japonicum can be fractionated into components which are almost completely high spin and almost completely low spin, respectively. The EPR and optical spectra of low spin forms are similar to those for low spin ferric cytochromes P-450 from other sources suggesting that in the low spin Rhizobium proteins at least one of the heme ligands is the same. The addition of phenobarbital to low spin Rhizobium cytochrome P-450 changes the optical spectrum ( λ max = 535,566nm) studied at room semperature to that of a high spin form ( λ max = 505,645 nm). Lowering the temperature to near 80 °K diminishes the spectral features of this high spin form while shifting the spectrum back to that of low spin protein. The low temperature (~1.6 °K) EPR spectrum of phenobarbitaltreated low spin cytochrome P-450 shows no new high spin ferric protein, while EPR absorptions ascribable to a low spin ferric phenobarbital bound form are observed. It is concluded that the spin state of phenobarbital-treated cytochrome P-450 is temperature dependent and this accounts for the absence of an EPR spectrum of high spin cytochrome P-450 in mammalian liver microsomes from phenobarbital-treated animals.