1. Volume 89, Issue 1, Pages 418-432 (July 2005)
Resolution of Two Substrate-Binding Sites in an Engineered Cytochrome P450eryF Bearing a Fluorescent Probe 
Dmitri R. Davydov, Alexandra E. Botchkareva, Nadezhda E. Davydova, James R. Halpert 
Biophysical Journal 
Volume 89, Issue 1, Pages (July 2005)
DOI: /biophysj
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2. Figure 1
1-PB binding to P450eryF mutants and CPM-labeled enzyme: wild-type (○), S93C (●), C154S (▿), C154A (▾), S93C/C154S (□), S93C/C154A (■), and CPM-labeled S93C/C154S mutant (P450eryF-CPM, ♢). Conditions: 0.1M Na-Hepes buffer, pH 7.4, 1mM DTT, 1mM EDTA, and 2μM enzyme, 25°C.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions

3. Figure 2
Effect of ionic strength on the high-spin content in the 1-PB-bound P450eryF wild-type, its mutants, and labeled derivatives. (a) Data obtained with the wild-type enzyme (○), S93C (●), C154S (▵), C154A (▴), S93C/C154S (□), and S93C/C154A (■). (b) Results of the experiments with S93C/C154S labeled with CPM (●), MDCC (■), and mBBr (♦). The curves characterizing P450eryF wild-type (○) and its unlabeled S93C/C154S mutant (□) are also shown in this panel for comparison. In these experiments, the ionic strength was adjusted by addition of 3M KCl in Hepes buffer. The concentration of 1-PB was 40μM. Other conditions were as in Fig. 1.
Biophysical Journal  , DOI: ( /biophysj )
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4. Figure 3
Cross correlation between the parameters of 1-PB binding and the effect of ionic strength on the spin equilibrium for a series of P450eryF variants with substitutions at position 154. (a) S50 versus Kion. (b) Hill coefficient, n, versus Kspin(0).
Biophysical Journal  , DOI: ( /biophysj )
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5. Figure 4
Determination of KD of the complex of labeled P450eryF S93C/C154S with 1-PB bound at the high-affinity binding site by FRET in a dilution setup. (a) Normalized emission spectra of an equimolar mixture of P450eryF with 1-PB at 0.23, 0.9, 1.4, 2.6, 3.4, 4.9, 7.6, 11, 13, and 16μM P450eryF-MDCC. Intensity of fluorescence is expressed in arbitrary units (a.u.). The inset shows the results of the same experiment as the plot of the amplitude of FRET given by the loading factor of the first principal component versus the concentration of P450eryF-MDCC. The solid line shows the approximation of this data set by Eq. 3 with KD=1.2μM. Conditions were as in Fig. 3. (b) The spectra of the first principal component derived from the experiments with equimolar mixtures of 1-PB with P450eryF-CPM (solid line), P450eryF-MDCC (long-dashed line), and P450eryF-bimane (short-dashed line).
Biophysical Journal  , DOI: ( /biophysj )
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6. Figure 5
Determination of KD of the complex of labeled P450eryF S93C/C154S with 1-PB bound at the low-affinity binding site by FRET in a dilution setup. (a) Normalized emission spectra of a mixture of P450eryF with 1-PB taken at a 1:3.3 molar ratio at 3.6, 4.7, 5.8, 6.8, 10, 12., 15, 17, 21, 23, and 25μM P450eryF-MDCC. Intensity of fluorescence is expressed in arbitrary units (a.u.). The inset shows the results of the same experiment as the plot of the amplitude of FRET given by the loading factor of the first principal component versus the concentration of P450eryF-MDCC. The solid line shows the approximation of this data set by the equation for the equilibrium of bimolecular association with KD=8.6μM. Conditions were as in Fig. 3. (b) The spectra of the first principal component derived from the experiments with the mixtures of 1-PB with P450eryF-MDCC taken at a molar ratio of 1:3.3 (dashed line) and 1:1 (solid line).
Biophysical Journal  , DOI: ( /biophysj )
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7. Figure 6
Changes in the spectra of absorbance of P450eryF-MDCC upon its interactions of 1-PB. (a) A series of the difference spectra derived from the titration of 12.7μM enzyme with 1-PB. The main panel shows the spectra obtained by subtraction of the spectrum of the substrate-free enzyme from the spectra measured at 2.9 (long-dashed line), 5.8 (medium-dashed line), 8.6 (short-dashed line), and 11μM (solid line) 1-PB. This experiment was done in a cell with an optical path length of 5mm. The inset shows the changes in the apparent concentration of the low-spin P450eryF derived from this experiment. The solid line represents the results of the fitting of this data set by Eq. 3 with KD=0.9μM. (b) A series of the difference spectra derived from the titration of 2.4μM enzyme with 1-PB. The main panel shows the spectra obtained by subtraction of the spectrum of the substrate-free enzyme from the spectra measured at 2.6 (solid line), 5.1 (long-dashed line), 7.2 (medium-dashed line), 10 (short-dashed line), 12 (dotted line),14 (dash-dotted line), 18 (dash-double-dotted line), and 23μM (solid line) 1-PB. The optical path length of the cell used in this experiment was equal to 10mm. The inset shows the changes in the high-spin fraction of the enzyme derived from this experiment. The solid line represents the results of the fitting of this data set by Eq. 5 (see Discussion) with KD1=1.2 and KD2=8.5μM.
Biophysical Journal  , DOI: ( /biophysj )
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8. Figure 7
Ionic-strength-induced changes in the content of the high-spin state of P450eryF-MDCC in the enzyme saturated with 1-PB (○), and in semisaturated (□) and substrate-free (♢) enzyme . The resulting curves were obtained by addition of KCl to a 2.6μM solution of P450eryF-MDCC with no substrate added (♢) and to the mixtures of P450eryF-MDCC with 1-PB taken at concentrations of 2.9 and 40μM (○) and 11 and 11μM, respectively. Solid lines represent the results of the fitting of these data sets by Eq. 1.
Biophysical Journal  , DOI: ( /biophysj )
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9. Figure 8
Models of part of the crystal structures of the complexes of P450eryF with 6-DEB (left) and ketoconazole (right) (28) showing putative charge-pairing contacts in the vicinity of Cys-154, the side chain of which is shown in yellow as a space-filling model. Space filling models also designate Ser-93 (magenta), heme (gray), and substrate (either 6-DEB or ketoconazole) (pale green). Red and green stick models show acidic and basic amino acid residues participating in salt links.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2005 The Biophysical Society Terms and Conditions