[Absorption and magnetic circular dichroism spectra of hemoproteins in nonequilibrium states. V. Cytochrome P450 and its substrate complex].
It was shown that ferrocytochrome P450 forms a nonequilibrium state if ferrocytochrome P450 and its complexes are reduced in freezed water-glycerol solutions by thermolysed electrons, arising during gamma-radiolysis of the matrix at 77 degrees K. Unlike the equilibrium form of ferrocytochrome P450 with the heme iron at the high-spin state the reduced nonequilibrium form of the protein contains the heme iron at a low-spin state. The absorption spectrum of ferrocytochrome P450 in the nonequilibrium state is characterized by alpha and beta-bands at 562 and 534 nm, respectively, whereas the magnetic circular dichroism spectra exhibit type A effect at 562 nm. Upon temperature increasing the nonequilibrium state is relaxed to the equilibrium one. Type 1 substrates had practically no influence on the spectral characteristic of the nonequilibrium form of ferrocytochrome P450. Binding of type 2 substrates results in an essential decrease of the intensity ratio of the alpha- and beta-bands (A alpha/A beta) and is accompanied by a red-shift of the alpha-band and corresponding magnetic circular dichroism effect. It was shown that mercaptoethanol complex of hemoglobin, formed by reduction at 77 degrees K is spectrally similar to the nonequilibrium ferrocytochrome P450 complex with type 2 substrates. From analysis of experimental data one can conclude that (i) the ligand environment of heme iron in oxidased and reduced cytochrome P450 are different; (ii) the sixth axial ligand of the heme iron in the oxidised protein is probably a water molecule (OH-) attached by a hydrogen bond to the neighbouring histidine. It is assumed that a similar nonequilibrium form of cytochrome P450 can be formed in physiological conditions.
D000818:Animals; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006418:Heme; D006420:Hemeproteins / Q000378:metabolism; D006860:Hydrogen Bonding; D007501:Iron; D008024:Ligands; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D013053:Spectrophotometry
D006420:Hemeproteins; D008024:Ligands; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D007501:Iron
Issledovanie spektrov pogloshcheniia i magnitnogo krugovogo dikhroizma gemoproteinov v konformatsionno neravnovesnykh sostoianiiakh. V. Tsitokhrom P450 i ego kompleksy s substratami.