Evidence for the existence of a high spin—low spin equilibrium in liver microsomal cytochrome <i>P</i>‐450

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

doi:10.1016/0014-5793(77)80043-4

Cited by 40 publications

(24 citation statements)

References 13 publications

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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: Resultssupporting

confidence: 84%

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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Section: Resultssupporting

confidence: 84%

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 finding that the C -0 stretching frequency of PB P-450-CO (uco 1949 cm-I) (Rein et al, 1977) is as high as that of HbCO (vco 1951 cm-I) (Alben and Caughey, 1968) is unexpected, because the change in band IV frequency upon the formation of CO complex [ A = v ( Fe'+-CO)u( Fe?+)] is fairly larger for P-450-CO ( A = 24 cm--l) than for HbCO ( A = I7 cm-1). This apparent inconsistency between A and uco might be interpreted reasonably if the CO-histidine (distal) interaction in flbCO (Ikeda-Saito et al, 1977) i s taken into account.…”

Section: Discussionmentioning

confidence: 96%

Resonance raman studies of hepatic microsomal cytochromes P-450: evidence for strong .pi. basicity of the fifth ligand in the reduced and carbonyl complex forms

Ozaki¹,

Kitagawa²,

Kyōgoku³

et al. 1978

Biochemistry

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Resonance Raman spectra have been measured for cytochromes P-450 purified from liver microsomes of phenobarbital-treated rabbits (PB P-450 and PB P-448) and of 3-methylcholanthrene-treated rabbits (MC P-448). In the reduced state, all three cytochromes P-450 exhibit Raman spectra of ferrous high-spin type but show the so-called "oxidation state marker" (band IV) at unusually low frequencies, indicating extensive delocalization of electrons from the iron dpi orbital to the porphyrin II (eg) orbital and, consequently, the strong pi basicity of the fifth ligand of the heme iron. The reduced CO complexes of the cytochromes P-450 also exhibit band IV at markedly lower frequencies than CO complexes of hemoglobin and myoglobin. These anomalies observed for the reduced form and CO complex disappear upon conversion of the cytochromes to the catalytically inactive form called cytochrome P-420. Oxidized PB P-450 shows a Raman spectrum which is characteristic of typical ferric low-spin heme compounds, whereas those of PB P-448 and MC P-448 are of the ferric high-spin type. PB P-450 is also clearly distinguishable from the two P-448 preparations in the reduced state. The reduced form of cytochrome P-420, produced by laser illumination, exhibits two sets of Raman lines and, therefore, seems to be a mixture of both high- and low-spin species.

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“…Results obtained through the efforts of his group provide a classic paradigm forming studies during “Biophysical and Biochemical Period” [2] which mapped the landscape and paths for future generations investigating this class of enzymes that play a central role in human health and disease [3]. These include application of spectroscopic methods for substrate binding and spin-shift thermodynamics and kinetics [4-6], studies of interaction with redox partners for hepatic and mitochondrial cytochromes P450 [7-8], cryogenic spectroscopy of binding intermediates and non-equilibrium states obtained by cryoradiolytic reduction and cryogenic photolysis [9-10], magneto-optical methods [11], novel spin labeled and fluorescent dye labeled components [12], and other methods of modern physical chemistry which have often led in developing new tools for biochemistry of membrane proteins.…”

Section: Introductionmentioning

confidence: 99%

Cytochromes P450 in Nanodiscs

Denisov

Sligar

2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators.

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Evidence for the existence of a high spin—low spin equilibrium in liver microsomal cytochrome P‐450

Cited by 40 publications

References 13 publications

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

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

Resonance raman studies of hepatic microsomal cytochromes P-450: evidence for strong .pi. basicity of the fifth ligand in the reduced and carbonyl complex forms

Cytochromes P450 in Nanodiscs

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