scholarly journals Domains of the catalytically self-sufficient cytochrome P-450 BM-3. Genetic construction, overexpression, purification and spectroscopic characterization

1992 ◽  
Vol 288 (2) ◽  
pp. 503-509 ◽  
Author(s):  
J S Miles ◽  
A W Munro ◽  
B N Rospendowski ◽  
W E Smith ◽  
J McKnight ◽  
...  
Keyword(s):  
Spin State ◽  
Equivalent Form ◽  
High Spin State ◽  
Spectroscopic Characterization ◽  
Hydroxylase Activity ◽  
Fatty Acid Hydroxylase ◽  
X Band ◽  
Resonance Raman Spectra ◽  
Cytochrome P 450 ◽  
Reductase Domain

1. The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli. 2. The protein products of these genes have been overexpressed and purified to homogeneity. 3. The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM). The cytochrome P-450 reductase domain readily reduces cytochrome c. Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3. 4. The X-band e.p.r. spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem. The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam. 5. Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given. Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments.

scholarly journals Structural alteration of mouse P450coh by mutation of glycine-207 to proline: spin equilibrium, enzyme kinetics, and heat sensitivity

1993 ◽  
Vol 294 (1) ◽  
pp. 31-34 ◽  
Author(s):  
R O Juvonen ◽  
M Iwasaki ◽  
T Sueyoshi ◽  
M Negishi
Keyword(s):  
Water Molecule ◽  
High Spin ◽  
High Spin State ◽  
Structural Alteration ◽  
Axial Position ◽  
Heat Sensitivity ◽  
Hydroxylase Activity ◽  
Coumarin 7 ◽  
Haem Protein ◽  
Cytochrome P 450

Mouse cytochrome P450coh is a high-spin haem protein which specifically catalyses coumarin 7-hydroxylase activity. A mutation of Gly-207 to Pro shifts the P450coh completely to the low-spin form, indicating that the sixth axial position of the haem is hexaco-ordinated with a water molecule in the mutant G207P. Moreover, the G207P mutation increases the Km value for coumarin 7-hydroxylase activity 100-fold and the Kd value for coumarin binding 200-fold. Conversely, the mutation decreases the Ki and Kd values 10- and 20-fold respectively when testosterone, a larger molecule, is used as a substrate. The results, therefore, are consistent with an idea that the substrate pocket may be larger in the mutant G207P than in the wild-type cytochrome P-450. A Gly-207 to Ala mutation (G207A) of P450coh (G207A), on the other hand, affects neither the spectral nor the enzymic properties of P450coh. Pro-207, through cis/trans isomerization or formation of a kink, may confer on the G207P a structural alteration of its substrate-haem pocket. Our previous studies [Iwasaki, Juvonen, Lindberg and Negishi (1991) J. Biol. Chem. 266, 3380-3382; Juvonen, Iwasaki and Negishi (1991) J. Biol. Chem. 266, 16431-16435] show that the residue at position 209 in P450coh resides close to the sixth axial position of the haem, and the spin equilibrium of the cytochrome P-450 shifts toward the high-spin state as residue 209 becomes more hydrophobic and larger. A Gly-207 to Pro mutation, therefore, results in the creation of a larger substrate pocket in the mutant cytochrome P-450 by altering the protein structure around residue 209 so that a water molecule and testosterone can be accommodated.

An Anomaly in the Resonance Raman Spectra of Cytochrome P-450cam in the Ferrous High-spin State

1976 ◽  
Vol 80 (6) ◽  
pp. 1447-1451 ◽  
Author(s):  
Yukihiro OZAKI ◽  
Teizo KITAGAWA ◽  
Yoshimasa KYOGOKU ◽  
Hideo SHIMADA ◽  
Tetsutaro IIZUKA ◽  
...  
Keyword(s):  
Raman Spectra ◽  
High Spin ◽  
Spin State ◽  
Resonance Raman ◽  
High Spin State ◽  
Resonance Raman Spectra

scholarly journals Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium

1997 ◽  
Vol 323 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Vincenzo LONGO ◽  
Giada AMATO ◽  
Annalisa SANTUCCI ◽  
Pier Giovanni GERVASI
Keyword(s):  
Spin State ◽  
High Spin State ◽  
Total Oxidation ◽  
Oxidation Rates ◽  
Sds Page ◽  
Molecular Masses ◽  
Endogenous Substrates ◽  
Cytochrome P 450 ◽  
Great Portion

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals.

Theoretical study on the reduction reactions from solid char(N): The effect of the nearby group and the high-spin state

Energy ◽  
2019 ◽  
Vol 189 ◽  
pp. 116286 ◽  
Author(s):  
Hai Zhang ◽  
Lei Luo ◽  
Jiaxun Liu ◽  
Anyao Jiao ◽  
Jianguo Liu ◽  
...  
Keyword(s):  
High Spin ◽  
Spin State ◽  
Theoretical Study ◽  
High Spin State ◽  
Reduction Reactions

scholarly journals Supercooled high spin state in metallo-supramolecular assemblies

2007 ◽  
Vol 63 (a1) ◽  
pp. s202-s202
Author(s):  
U. Pietsch ◽  
M. Lommel ◽  
Y. Bodethin ◽  
D. Kurth ◽  
G. Schwarzl ◽  
...  
Keyword(s):  
High Spin ◽  
Spin State ◽  
High Spin State ◽  
Supramolecular Assemblies

scholarly journals Spin Crossover in Nickel(II) Tetraphenylporphyrinate via Forced Axial Coordination at the Air/Water Interface

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4155
Author(s):  
Alexander V. Shokurov ◽  
Daria S. Kutsybala ◽  
Andrey P. Kroitor ◽  
Alexander A. Dmitrienko ◽  
Alexander G. Martynov ◽  
...  
Keyword(s):  
Spin State ◽  
Spin Crossover ◽  
High Spin State ◽  
Water Interface ◽  
Metal Centers ◽  
Axial Coordination ◽  
Vis Spectroscopy ◽  
Air Water Interface ◽  
Nickel Cation

Coordination-induced spin crossover (CISCO) in nickel(II) porphyrinates is an intriguing phenomenon that is interesting from both fundamental and practical standpoints. However, in most cases, realization of this effect requires extensive synthetic protocols or extreme concentrations of extra-ligands. Herein we show that CISCO effect can be prompted for the commonly available nickel(II) tetraphenylporphyrinate, NiTPP, upon deposition of this complex at the air/water interface together with a ruthenium(II) phthalocyaninate, CRPcRu(pyz)2, bearing two axial pyrazine ligands. The latter was used as a molecular guiderail to align Ni···Ru···Ni metal centers for pyrazine coordination upon lateral compression of the system, which helps bring the two macrocycles closer together and forces the formation of Ni–pyz bonds. The fact of Ni(II) porphyrinate switching from low- to high-spin state upon acquiring additional ligands can be conveniently observed in situ via reflection-absorption UV-vis spectroscopy. The reversible nature of this interaction allows for dissociation of Ni–pyz bonds, and thus, change of nickel cation spin state, upon expansion of the monolayer.

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