Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures.

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure.

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.

Electron paramagnetic resonance investigation of cytochrome P-450c21 from bovine adrenocortical microsomes: a new enzymatic activity.

Cytochrome P-450c21 (CYP21A1) purified from bovine adrenocortical microsomes was investigated by electron paramagnetic resonance (EPR) spectroscopy to clarify the interactions among heme active center, protein surroundings, water molecules and bound substrates or analogues. The low-spin EPR signals of the oxidized enzyme (as purified) consisted of two species; one at gz = 2.39, gy = 2.23, and gx = 1.925 (component A) and the other at gz = 2.42, gy = 2.23, and gx = 1.916 (component B). The component A is probably representing a product-bound form, whereas the component B indicates either occupation of the substrate-binding site with a substrate analogue or absence of steroid at the site. Upon addition of progesterone, the component A signal completely disappeared and the intensity of high-spin signal (g = 8.06, 3.54) increased. Addition of 17 alpha-hydroxyprogesterone caused a development of a new low-spin signal at gz = 2.42, gy = 2.21, and gx = 1.966 (component C) and a further increase in intensity of the high-spin signal (g = 8.06, 3.54). Addition of 20 beta-hydroxyprogesterone caused an increase in intensity of the component C signal (and the g = 8 high-spin signal) even stronger than did 17 alpha-hydroxyprogesterone. These observations suggest that 20 beta-hydroxyprogesterone binds to the cytochrome P-450c21 active center in a very similar manner as 17 alpha-hydroxyprogesterone does and, therefore, may be a metabolizable substrate. A new enzymatic pathway catalyzed by cytochrome P-450c21 was confirmed with a reconstituted enzymatic system consisting of cytochrome P-450c21, NADPH-cytochrome P-450 reductase and an NADPH-generating system. 20 beta-Hydroxyprogesterone was converted to progesterone via a putative 20 beta-oxidase reaction in a comparable turnover number to that of the 21-hydroxylation of 17 alpha-hydroxyprogesterone.

EPR studies on the photoproducts of ferric cytochrome P450cam (CYP101) nitrosyl complexes: effects of camphor and its analogues on ligand-bound structures.

Photolyzed products of the NO complexes of ferric cytochrome P450cam both in the substrate-free and several substrate-bound states were trapped and examined by EPR spectroscopy at 5 K. In the absence of substrate, the photoproduct exhibited ferric high- and low-spin signals, neither of which showed the line-width broadening characteristic of magnetic interaction between photodissociated NO and the heme iron. This finding indicates that photodissociated NO can diffuse to the unconstrained distal heme pocket giving the high-spin heme, and that a part of the high-spin heme species converts to the low-spin heme upon coordinating an aqua molecule. When a substrate, camphor or adamantanone, was bound at the site above the heme, the photoproduct exhibited widespread EPR absorptions together with a new distinct signal at g approximately 4.4. The new signals are assignable to a weakly spin-coupled species between the ferric heme iron and the photodissociated NO, indicating that the NO molecule is in close proximity to the heme iron by the steric crowding of the bound substrate. The photoproduct of the norcamphor complex exhibited a spin-coupled EPR signal at g approximately 5, in which the coupling is suggested to be weaker than that of the camphor-bound enzyme. On the other hand, the photoproduct of the NO complex in an adamantane-bound state only yielded low-spin signals, and exhibited no spin-coupled signals. This result suggests that adamantane is mobile in the substrate pocket due to the lack of hydrogen bond formation with Tyr96.(ABSTRACT TRUNCATED AT 250 WORDS)

EPR studies on the photoproducts of manganese(II) protoporphyrin-IX substituted myoglobin nitrosyl complexes trapped at low temperature: effects of site-specific chemical modification of the distal histidine on ligand-binding structures.

Manganese(II) protoporphyrin-IX substituted myoglobin with site-specifically cyanated or N-tetrazolated distal histidine (His) was prepared and low-temperature photolysis of nitric oxide (NO) from their nitrosyl complexes was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the steric crowding of the distal heme moiety. The photoproduct of NO complex of the tetrazolated Mn(II)Mb (tetrazole-Mn(II)Mb) exhibited widespread absorption in the magnetic field from zero to 0.4 T due to a spin-coupled interaction between the high-spin Mn(II) center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. This indicates that the NO complex of tetrazole-Mn(II)Mb has sterically restricted distal heme pocket. On the other hand, the photoproduct of NO complex of cyanated Mn(II)Mb (BrCN-Mn(II)Mb) exhibited only the broad g = 6 absorption due to the magnetic dipole-dipole interaction between the photodissociated NO and the high-spin Mn(II) center. A drastic conformational change in the heme-ligand moiety, in which the distal histidine side chain is pushed toward the outside of the heme pocket, leaving an open space in the distal heme pocket, can be suggested.

Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.