Quantitative analysis of the spin equilibrium of cytochrome LM-2 fraction from rabbit liver microsomes

Ristau, O.; Rein, H.; Jänig, G.-R.; Ruckpaul, K

doi:10.1016/0005-2795(78)90068-5

Cited by 46 publications

(17 citation statements)

References 21 publications

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“…Docking of excess benzphetamine to the predominantly low-spin protein caused conversion to an approximately equal mixture of high-and low-spin forms [128], with the high-spin contribution increasing from about 17 to 48% as the temperature was gradually elevated [129]. Efforts were made to scrutinize the thermodynamic basis of the substrate-induced change in spin equilibrium of the heme iron [130], and experiments with a set of benzphetamine analogues in fact hinted at a linear dependence on each other of the free energies of substrate binding (K d ) and stabilization of the substrate-triggered high-spin state [131].…”

Section: The Cyp2b Enzymesmentioning

confidence: 98%

“…Finally, attention has to be drawn to the experimental setup designed to assess the impact of substrate on spin equilibria. As is known, spin transition in P450s is highly prone to thermal effects [32,43,72,129,167], so that proper conclusions as to spin-state changes at physiological temperatures might be impeded by the use of information from Mössbauer [12] and EPR [11,16,98,116,157,179] spectral measurements carried out under cryogenic conditions. However, this possibility seems to be of minor importance, since careful examination revealed more than 80% of the spectral data discussed in the present review to originate from analyses by UV-visible absorption spectroscopy performed at ambient temperatures.…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

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Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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Based on initial studies with bacterial CYP101A1, a popular concept emerged predicting that substrate-induced low-to-high spin conversion of P450s is universally associated with shifts of the midpoint potential to a more positive value to maximize rates of electron transfer and metabolic turnover. However, evaluation of the plethora of observations with pro- and eukaryotic hemoproteins suggests a caveat as to generalization of this principle. Thus, some P450s are inherently high-spin, so that there is no need for a supportive substrate-triggered impulse to electron flow. With other enzymes, high-spin content is not consonant with reductive activity, and spin transition as such is not essential to sustaining substrate oxidation. Also, with certain proteins the low-spin conformer is reduced as swift as the high-spin entity. Moreover, there is not regularly a linear relationship between high-spin level and anodic shift of the reduction potential. Similarly, in given cases turnover may proceed despite insignificant or even lacking substrate-provoked alterations in the redox behaviour. Thus, folding of the disparate and sometimes conflicting data into a harmonized overall picture is a lingering problem. Apart from direct perturbation of the electrochemical properties, substrate docking may entail changes in enzyme conformation such as to favour productive complexation with redox partners or modulate electron transfer conduits within preformed donor/acceptor adducts, resulting in elevated ease of flow of reducing equivalents. Substrate-steered ordering of the oligomeric aggregation state of P450s is likely to impose steric constraints on heterodimers, causing one component to more readily align with electron carriers. Careful uncovering of electrochemical mechanisms in these systems will be fruitful to tailoring of novel bioenergetic machines and redox chains via redox-inspired protein engineering or molecular Lego, capable of generating products of interest or degrading toxic pollutants. Finally, availability of P450 nanobiochips for high-throughput screening of substrate libraries might expedite drug development.

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Section: The Cyp2b Enzymesmentioning

confidence: 98%

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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“…This initially puzzling result may be due to the fact that MF and BP binding to CYP2B4 is mediated largely by hydrophobic contributions to the total binding free energy that are smaller at lower temperatures 47. The results in Figure 1 are in agreement with previous results, which demonstrated that BP binds with greater affinity at higher temperatures and that that binding was predominantly an entropy driven process 48, 49…”

Section: Resultsmentioning

confidence: 99%

“…Comparison of the electrostatic versus hydrophobic/apolar analysis components in Table IV(C) reveals that the hydrophobic/apolar binding components become less favorable by 6.5 kcal/mol at low temperature, while the polar/electrostatic terms are virtually unchanged. These results for BP are consistent with the interpretations of early van Hoft analysis 48, 49…”

Section: Resultsmentioning

confidence: 99%

“…47 The results in Figure 1 are in agreement with previous results, which demonstrated that BP binds with greater affinity at higher temperatures and that that binding was predominantly an entropy driven process. 48,49 Low-Energy Conformations of MF and BP Figure 2 shows low-energy conformations of MF and BP determined from molecular mechanics assessments using two distinct force fields. Both the global energy minimum and the energy ordering of conformers on the molecular mechanics potential surface of both ligands was the same irrespective of whether CHARMM 50 or AMBER 5 30 potential functions were employed.…”

Section: Results and Discussion Determination Of The Spectral Dissocimentioning

confidence: 99%

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Theoretical study of the ligand–CYP2B4 complexes: Effect of structure on binding free energies and heme spin state

et al. 2004

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The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.

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Physicochemical Properties of Multiple Forms of Cytochrome P-450: A Proposal for the Ligand Structure

Yoshida¹

1985

P-450 and Chemical Carcinogenesis

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Quantitative analysis of the spin equilibrium of cytochrome LM-2 fraction from rabbit liver microsomes

Cited by 46 publications

References 21 publications

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Theoretical study of the ligand–CYP2B4 complexes: Effect of structure on binding free energies and heme spin state

Physicochemical Properties of Multiple Forms of Cytochrome P-450: A Proposal for the Ligand Structure

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