The high-spin/low-spin equilibrium in cytochrome P-450 — A new method for determination of the high-spin content

Jung, Christiane; Ristau, O.; Rein, H.

doi:10.1016/0167-4838(91)90229-s

Cited by 44 publications

(39 citation statements)

References 19 publications

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“…Absorbance spectroscopy has been used previously to monitor the change in spin-state in both CYPs [15,37,40,[42][43][44][45]. Ligand-induced absorbance changes of heme absorbance bands of CYPs can be monitored by a variety of methods, including: simple absorbance difference, normalized absorbance difference, second derivative analysis, curve fitting, and the direct measurement of the apparent absorbance bands [15,37,40,[42][43][44][45].…”

Section: Resultsmentioning

confidence: 99%

“…Absorbance spectra were deconvoluted into their LS and HS components and the broad δ-band using the multiple peak fitting package of Igor Pro 5.0 (Wavemetrics, Inc., Lake Oswego, Oregon) in a similar fashion as described for P450 CAM , P450 eryF and CYP3A4 [15,40,41]. The relative areas under these peaks were previously calculated and compared to a low spin (LS) reference spectra to estimate percentages of LS and HS [15,41].…”

Section: Estimation Of Hs and Ls By Absorbance Spectroscopymentioning

confidence: 99%

“…simple absorbance difference, second derivative analysis, curve fitting, or direct measurement of the apparent absorbance bands [40,[42][43][44][45]) because it is not dependent on the extinction coefficients and it is insensitive to protein concentration and solvent artifacts. However, line width of Soret bands are not taken into account and the Soret bands are assumed to have similar extinction coefficients.…”

Section: Normalized Absorbance Difference Spectroscopymentioning

confidence: 99%

“…Curve fitting has been used previously to determine the %LS and %HS in CYPs [15,40,41]. Determining the fraction of LS and HS can provide insight into the coupling between spin state and ligand binding [15].…”

Section: Estimation Of Low Spin and High Spin Concentration Of Cyp3a4mentioning

confidence: 99%

“…The HS and LS Soret bands are obviously associated with the HS and LS heme, respectively, while the δ-band has not been ascribed to a specific property of the heme in the literature. These bands can be deconvoluted by curve fitting as previously described [15,40]. From the areas of the Soret peaks, the relative concentration of HS and LS can be calculated with respect to suitable controls.…”

Section: Estimation Of Low Spin and High Spin Concentration Of Cyp3a4mentioning

confidence: 99%

See 4 more Smart Citations

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Roberts¹,

Atkins²

2007

Archives of Biochemistry and Biophysics

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Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, α-naphthoflavone (ANF) and the steroid, testosterone (TST). UV-vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: 1) a relatively tight spin-state insensitive binding site (CYP•ANF) and 2) a relatively low affinity spin-state sensitive binding site (CYP•ANF•ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s).

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Section: Resultsmentioning

confidence: 99%

Section: Estimation Of Hs and Ls By Absorbance Spectroscopymentioning

confidence: 99%

Section: Normalized Absorbance Difference Spectroscopymentioning

confidence: 99%

Section: Estimation Of Low Spin and High Spin Concentration Of Cyp3a4mentioning

confidence: 99%

Section: Estimation Of Low Spin and High Spin Concentration Of Cyp3a4mentioning

confidence: 99%

See 3 more Smart Citations

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Roberts¹,

Atkins²

2007

Archives of Biochemistry and Biophysics

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Steroid Hydroxylation: Microbial Steroid Biotransformations Using Cytochrome P450 Enzymes

Bureik

Bernhardt

2007

Modern Biooxidation

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Novel family members of CYP109 from Sorangium cellulosum So ce56 exhibit characteristic biochemical and biophysical properties

Khatri

Hannemann

Girhard³

et al. 2013

Biotech and App Biochem

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The members of the CYP109 family (CYP109C1, CYP109C2, and CYP109D1) from Sorangium cellulosum So ce56 are among the 21 P450 enzymes, of which only CYP109D1 and CYP264B1 have so far been functionally characterized. Here, we attempted to characterize two other P450s (CYP109C1 and CYP109C2) for the first time and compare their biochemical, biophysical, and functional properties to those of the fatty acid hydroxylating CYP109D1. Considering the physiological importance of fatty acids, we investigated saturated fatty acid binding and conversion for all members of the CYP109 family. The interaction between the CYP109 members and different autologous/heterologous redox partners was compared using Biacore measurements in which only CYP109D1 and bovine adrenodoxin (Adx) formed a complex. Surprisingly, this interaction was similarly efficient as the interaction of Adx with its mammalian redox partners. The in vitro reconstitution assays showed no activity when using CYP109C1, although substrate binding was demonstrated; also, there was subterminal hydroxylation of saturated fatty acids, when using CYP109C2 and CYP109D1, where CYP109D1 was a much more efficient fatty acid hydroxylase. Interestingly, the hydroxylation position moved inside the fatty acid chain when using long-chain fatty acids, thus producing possible precursors for physiologically important products.

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The high-spin/low-spin equilibrium in cytochrome P-450 — A new method for determination of the high-spin content

Cited by 44 publications

References 19 publications

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

Steroid Hydroxylation: Microbial Steroid Biotransformations Using Cytochrome P450 Enzymes

Novel family members of CYP109 from Sorangium cellulosum So ce56 exhibit characteristic biochemical and biophysical properties

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