©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
X-ray Absorption Near Edge Studies of Cytochrome P-450-CAM, Chloroperoxidase, and Myoglobin
DIRECT EVIDENCE FOR THE ELECTRON RELEASING CHARACTER OF A CYSTEINE THIOLATE PROXIMAL LIGAND (*)

Hongbin Isaac Liu (1), Masanori Sono (3), Saloumeh Kadkhodayan (3), Lowell P. Hager (5), Britt Hedman (2), Keith O. Hodgson (1) (2), John H. Dawson (3) (4)

From the (1) Department of Chemistry and the (2) Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, California 94305, the (3) Department of Chemistry and Biochemistry and the (4) School of Medicine, University of South Carolina, Columbia, South Carolina 29208, and the (5) Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The low spin ferric and low and high spin ferrous forms of myoglobin, bacterial cytochrome P-450-CAM, and chloroperoxidase have been examined by Fe-K x-ray absorption edge spectroscopy. The positions of the absorption edge and the shapes of preedge and edge regions of imidazole adducts of ferric P-450-CAM and chloroperoxidase are essentially the same when compared with thiolate-ligated ferric myoglobin. As these three protein derivatives all have six-coordinate, low spin, ferric hemes with axial imidazole and thiolate ligands, the superposition of x-ray absorption edge spectral properties demonstrates that the protein environment does not effect the spectra, provided one compares heme iron centers with identical coordination numbers, spin and oxidation states, and ligand sets. In contrast, a 0.96 eV difference is observed in the energy of the absorption edge for imidazole- and thiolate-ligated ferric myoglobin with the latter shifted to lower energy as observed for ferrous myoglobin states. Similarly, in the low spin ferric-imidazole and ferrous-CO states, the energies of the absorption edge for chloroperoxidase and P-450-CAM are shifted in the direction of the ferrous state (to lower energy) when compared with those for analogous myoglobin derivatives. In the deoxyferrous high spin state, comparison of the edge spectra of chloroperoxidase with analogous data for cytochrome P-450-CAM suggests that the electron density at the iron is similar for these two protein states. The shifts observed in the energies of the x-ray absorption edge for the thiolate-ligated states of these proteins relative to derivatives lacking a thiolate ligand provide a direct measure of the electron releasing character of a thiolate axial ligand. These results therefore support the suggested role of the cysteinate proximal ligand of P-450 as a strong internal electron donor to promote O-O bond cleavage in the putative ferric-peroxide intermediate to generate the proposed ferryl-oxo ``active oxygen'' state of the reaction cycle.


INTRODUCTION

Since molecular structure dictates the spectroscopic and catalytic properties of metalloenzyme active sites, a thorough understanding of the unique spectral features of cytochrome P-450 and chloroperoxidase has long been sought to provide insight into the mechanism of action of these two heme enzymes (1, 2) . P-450 and chloroperoxidase are unusual among heme enzymes in each having a cysteine thiolate proximal ligand. With P-450, it has been proposed that the cysteinate ligand plays a key mechanistic role as a strong internal electron donor to facilitate cleavage of the O-O bond of the putative iron-peroxide intermediate to generate the active hydroxylation catalyst (1, 2, 3, 4) . Using Fe-K x-ray absorption edge spectroscopy, we have probed the properties of the central metal of these two enzymes and of myoglobin, which has a histidine proximal ligand. Shifts in the energy of the x-ray absorption edge are observed that correlate with the presence of the thiolate proximal ligand. The results described herein provide the most direct evidence to date for the specific influence of the cysteinate ligand on the properties and possibly on the reactivities of the heme iron centers of cytochrome P-450 and chloroperoxidase.

Cytochrome P-450 is a ubiquitous heme iron monooxygenase found in bacteria, mammals, and plants (1, 2, 5, 6, 7) . Its ability to activate one atom in dioxygen for insertion into unactivated C-H bonds, with concomitant reduction of the other oxygen atom to water (Reaction 1), has generated substantial interest in its mechanism of action. The name P-450 was derived from the unusually red-shifted Soret absorption band at 450 nm for the ferrous-CO derivative of the enzyme. This property is clearly dependent on the presence of a cysteinate thiolate proximal heme iron ligand (Fig. 1); the role that ligand may play in the mechanism of the enzyme is less well established. P-450-CAM, obtained from Pseudomonas putida grown on (1 R)-camphor, catalyzes the stereo- and regio-specific hydroxylation of camphor to the 5- exo alcohol (6) .

On-line formulae not verified for accuracy

REACTION 1


Figure 1: Schematic representation of the active site heme iron coordination structures of cytochrome P-450 ( P-450) and chloroperoxidase ( CPO) ( left) and of myoglobin ( right) showing the endogenous proximal ligands and exchangeable distal sites.



Chloroperoxidase is an unusual heme enzyme isolated from the fungus Caldariomyces fumago(1, 2, 8, 9) that functions as a peroxidase (Reaction 2), catalase (Reaction 3), or halogenation catalyst (Reaction 4). Well documented spectroscopic similarities to P-450 support the conclusion that chloroperoxidase also has a cysteinate proximal ligand (Fig. 1). That chloroperoxidase, in contrast to P-450, is a peroxide-dependent halogenation catalyst and is incapable of dioxygen activation has focused attention on what must be significantly different structure-function relationships for the two thiolate-ligated heme enzymes.

On-line formulae not verified for accuracy

REACTION 2

On-line formulae not verified for accuracy

REACTION 3

On-line formulae not verified for accuracy

REACTION 4

The myoglobins are monomeric heme-containing oxygen carriers found in the vertebrate muscle tissue. The proximal ligand to the heme iron of the myoglobins is histidine (10) (Fig. 1). The ability of the myoglobins (and hemoglobins) to bind dioxygen reversibly has led to intense scrutiny by x-ray crystallography as well as spectroscopy.

X-ray absorption spectroscopy using synchrotron radiation is a powerful technique for high resolution studies of transition metal active sites in biological molecules because it directly probes the properties of the central metal (11, 12, 13) . The heme iron coordination structures of a variety of heme proteins have been studied by means of extended x-ray absorption fine structure (EXAFS)() spectroscopy (14, 15, 16, 17, 18, 19, 20) . Using this technique, it is possible to determine Fe-N and Fe- X bond distances to an accuracy of about ± 0.02 Å. We have previously characterized four P-450 states and two chloroperoxidase derivatives with this technique (21, 22, 23, 24) . X-ray crystal structures have been reported for three of the four P-450 states to have been examined by EXAFS (25, 26, 27) . The metal-ligand bond distances obtained with these two methods are in close agreement (13) . As usually applied in the analysis of first coordination shells for solution samples using empirical phase and amplitude functions, EXAFS is insensitive to the three-dimensional arrangement of atoms. The x-ray absorption edge spectrum, on the other hand, contains information about the oxidation state, structure, and coordination geometry of the heme iron (28, 29, 30, 31, 32) . We have systematically examined parallel derivatives of the three proteins, making careful comparison of the energy and shape of the absorption edge under conditions where the oxidation state, coordination number, and spin state are kept constant within a particular group being compared. In this way, the specific effect of the thiolate ligand that is present in P-450 and chloroperoxidase on the properties of the central heme iron has been quantified.


EXPERIMENTAL PROCEDURES

Cytochrome P-450-CAM was purified from Pseudomonas putida grown on (1 R)-camphor by the method of Peterson et al.(33) with minor modifications (34) . Chloroperoxidase was purified from C. fumago grown on fructose (35) as reported previously (36, 37) . Horse heart myoglobin (Sigma) was purified as described by Dawson et al.(38) . All chemicals were obtained from Sigma or Aldrich and were used as received. The derivatives of P-450, chloroperoxidase, and myoglobin were prepared at 4 °C in 75 mM potassium phosphate buffer (pH 7.0, 6.0, and 7.0, respectively) containing 25% (v/v) ethylene glycol to final protein concentrations of 2.5 mM. The ligand complexes of the ferric proteins were generated by adding the neat ligand ( N-methylimidazole) or concentrated ligand stock solutions in ethanol (1-propanethiol, p-chlorothiophenol, 4-phenylimidazole) or water (imidazole with pH adjustment) to the proteins. The formation and the homogeneity of the complexes were monitored by electronic absorption spectral changes of the peak at 630-640 nm of the native ferric proteins as well as by electron paramagnetic resonance spectroscopy at 77 K for the low spin ligand complexes. The deoxy-ferrous derivatives of P-450, chloroperoxidase, and myoglobin were prepared under N in a 2-mm cuvette by first reducing the ferric proteins (without ethylene glycol) with a few grains of solid sodium dithionite and then adding anaerobic ethylene glycol ( volume). Ferrous-CO derivatives were then prepared by gently bubbling with CO. Reduction and CO complex formation were again monitored by optical absorption spectroscopy between 600 and 700 nm. The samples were transferred to lucite cells with Kapton windows using an gas-tight syringe under air (ferric samples) or N (ferrous samples) and were stored at 77 K. The homogeneity and integrity of the protein before and after the x-ray absorption measurements were verified by electron paramagnetic resonance (for the ferric samples directly in the lucite cell) or by optical absorption spectroscopy (for anaerobically diluted ferrous samples in the presence or absence of CO, and for the diluted, ferrous-CO form of the ferric samples of P-450 and chloroperoxidase). These examinations revealed that no autoxidation of the ferrous samples had occurred and that less than 5% protein denaturation took place during sample manipulation or irradiation.

X-ray absorption spectra were measured between 10 and 85 K using an Oxford Instruments continuous flow liquid helium cryostat at the Stanford Synchrotron Radiation Laboratory (SSRL) and the National Synchrotron Light Source (NSLS). At SSRL, the data were collected as fluorescence excitation spectra on unfocused wiggler beam lines 7-3 and 4-2 using a Si (220) double crystal monochromator detuned 50% at 7474 eV for harmonic rejection. At NSLS, the data were collected on unfocused bending magnet beam line X19A using a Si (220) double crystal monochromator. A Canberra 13-element solid state germanium array detector was used in all experiments except one set at SSRL in which an ionization chamber detector of the Stern-Lytle design was used. The SSRL storage ring was operated under dedicated condition at 3.0 GeV and 35-95 mA. The NSLS storage ring was operated at 2.5 GeV and 90-200 mA.

Data reduction and analysis were performed with the XFPAKG program package (39, 40) . Energy calibration was done using an internal iron foil standard by assigning the first inflection point of the iron absorption edge at 7111.2 eV. The calibration was fine tuned by individually comparing and recalibrating each foil scan relative to a ``master'' scan until a reproducibility of <0.06 eV was obtained. Calibrated scans were further inspected individually for noise-caused inconsistencies, and unacceptable scans were identified and rejected during further data reduction. A weighted average of the remaining scans was calculated. The first and the last scans were compared and showed that no detectable photoreduction occurred for any of the samples. A preedge subtraction was performed by fitting the postedge region with a smooth polynomial, which was extrapolated into the preedge region and subtracted. A one-segment spline was fit to the postedge region and subtracted, at which point the data were normalized to an edge jump of one.

The edge separation was quantified in three separate ways. The edges were fit using the program EDG_FIT (written by Dr. G. N. George, SSRL), which utilizes the double precision version of the public domain MINPAK fitting library (Garbow, B. S., Hillstrom, K. E., and More, J. J., Argonne National Laboratory). All spectra were fit over the range 7100-7190 eV. The preedge and all edge features including the rising edge were all modeled by pseudo-Voigt line shapes (sums of Gaussian and Lorenzian functions). A fixed ratio (1:1) of Gaussian/Lorenzian were used for all functions. The number of functions and their initial energy positions were determined from the second derivative of the spectrum. An arctangent step function was applied to model the total edge jump. The number of functions were minimized and kept constant for samples of each group, such as N-methylimidazole-ligated chloroperoxidase, N-methylimidazole-ligated P-450, and 1-propanethiol-ligated myoglobin. In the fits, the line-width, energy position, and height were allowed to vary in a stepwise manner. The difference in edge position was then calculated as the difference between the energy of the maximum of the first highest transition for each edge. The uncertainty in the determination is estimated to be 0.1 eV. The edge separation was also estimated from the difference in the second derivative at the inflection point of the highest rising edge transition and by calculating an average energy difference for a number of points at the same height for the highest rising edge transition for samples under comparison, giving results consistent with those from the fits above.


RESULTS AND DISCUSSION

Rationale

The rationale of the present study is that the energy and shape of the x-ray absorption edge for two heme iron protein derivatives should be the same if the two iron centers have identical oxidation and spin states and matching axial ligands. Once that premise is established, it should then be possible to change one of the ligands while keeping the oxidation and spin states constant in order to specifically probe the electron donor properties of that ligand. This approach has been used in the present study to directly examine the effect a thiolate sulfur donor axial ligand has on the energy and shape of the x-ray absorption edge relative to the influence of a histidine nitrogen donor ligand.

To test the rationale, the edge spectra of the N-methylimidazole adducts of ferric cytochrome P-450-CAM and chloroperoxidase were compared to that of a 1-propanethiolate adduct of ferric myoglobin (Fig. 2, top). All three samples have imidazole and thiolate axial ligands and a six-coordinate, low spin ferric iron. The shape and energy of the absorption edge are essentially indistinguishable among the three spectra. This conclusion is further supported in the difference x-ray absorption spectra in the bottom of Fig. 2. In separate experiments, it was found that the edge energies of ferric myoglobin complexed with either N-methylimidazole, 4-methylimidazole, or unsubstituted imidazole were identical or very similar (; data not shown); N-methylimidazole was used for subsequent experiments. A similar experiment comparing the edge energy for complexes of ferric myoglobin with aliphatic (1-propanethiol) and aromatic ( p-chlorothiophenol) thiolate ligands revealed a noticeable difference of 0.31 ± 0.03 eV in the edge energy (; spectrum not shown); because cysteine is an aliphatic thiol, 1-propanethiol was used in subsequent experiments.


Figure 2:Top, comparison of the Fe-K x-ray absorption edge spectra of the N-methylimidazole adducts of ferric cytochrome P-450-CAM ( N- P- 450, dottedline) and chloroperoxidase ( N- CPO, solidline) and the 1-propanethiolate complex of ferric myoglobin ( S-myoglobin, dashedline). Bottom, difference x-ray absorption spectra between S-myoglobin ( S- Mb) and N-CPO ( solidline) and between S-Mb and N-P-450 ( dottedline).



Having established that heme iron complexes with identical oxidation and spin states and axial ligand sets have nearly indistinguishable x-ray absorption edge spectra, the effect of changing one of the axial ligands was then examined. The edge spectra of the N-methylimidazole and 1-propanethiolate adducts of ferric myoglobin are shown in the top of Fig. 3. A clear shift of 0.96 eV to lower energy is observed in the energy of the rising edge for the thiolate-bound case relative to the imidazole adduct. The shift is especially evident in the difference spectrum displayed in the bottom of Fig. 3. A shift to lower energy is typically seen in the edge upon reduction of ferric heme iron complexes. This experiment thus demonstrates that the energy of the x-ray absorption edge is sensitive to the presence of a thiolate axial ligand. Furthermore, since the shift in the edge energy is to lower energy upon thiolate binding ( i.e. the same as for reduction of the ferric heme iron), this suggests that the thiolate ligand serves as a stronger electron donor to the iron than does an imidazole ligand.


Figure 3:Top, comparison of the Fe-K x-ray absorption edge spectra of 1-propanethiolate ferric myoglobin ( S-myoglobin) ( dottedline) and N-methylimidazole ferric myoglobin ( N-myoglobin) ( solidline). Bottom, difference x-ray absorption spectrum between N-myoglobin ( N- Mb) and S-myoglobin ( S- Mb).



Low Spin Ferric State

The x-ray absorption edge spectra of the N-methylimidazole adducts of ferric cytochrome P-450-CAM, chloroperoxidase, and myoglobin are displayed in the top of Fig. 4. The edge energies for the P-450-CAM and chloroperoxidase samples occur at lower energy than that of the myoglobin complex. Since the only difference among imidazole-ligated ferric P-450, chloroperoxidase, and myoglobin is the presence of a cysteine thiolate axial ligand in P-450 and chloroperoxidase compared with the histidine imidazole ligand for myoglobin, it is concluded that the edge shift seen in Fig. 4is indicative that the thiolate sulfur of cysteinate is a stronger electron donor than the imidazole nitrogen of histidine. The edge differences, as displayed in the bottom of the figure, are very similar in magnitude (1 eV) to that seen when comparing imidazole- and thiolate-ligated myoglobin (0.96 eV, Fig. 3), indicating strong similarity in the electronic and geometric environment among imidazole-ligated ferric P-450 and chloroperoxidase, and thiolate-ligated ferric myoglobin.


Figure 4:Top, comparison of the Fe-K x-ray absorption edge spectra of the N-methylimidazole adducts of ferric cytochrome P-450-CAM ( N- P- 450, dottedline), chloroperoxidase ( N- CPO, dashedline), and myoglobin ( N- Mb, solidline). Bottom, difference x-ray absorption spectra between N-Mb and N-CPO ( solidline) and between N-Mb and N-P-450 ( dottedline).



Furthermore the preedge and edge shapes, and energy positions of N-methyl-imidazole-chloroperoxidase and N-methylimidazole-P-450-CAM are very similar to those of 1-propanethiolate-myoglobin. Since myoglobin has a distal histidine imidazole, as established by x-ray crystallography and optical methods, the x-ray absorption edge spectra reflect the similarities of the electron donating properties of the thiolate ligand in the N-methylimidazole adducts of chloroperoxidase and P-450-CAM, and 1-propanethiolate-ligated myoglobin.

Low Spin Ferrous State

The edge spectra of low spin ferrous-CO myoglobin, chloroperoxidase, and P-450-CAM are shown in Fig. 5 . The energies of the edges for ferrous-CO chloroperoxidase and P-450-CAM occur at lower energy than for the corresponding myoglobin analog. This is consistent with the energy differences seen for the low spin ferric state and again demonstrates the greater electron donating effect of the thiolate ligand relative to an imidazole ligand on the electronic properties of the iron in low spin ferrous-CO chloroperoxidase and P-450-CAM.


Figure 5: Comparison of the Fe-K x-ray absorption edge spectra of the ferrous-CO derivatives of cytochrome P-450-CAM ( P-450, dash-dot line), chloroperoxidase ( CPO, dotted line) and myoglobin ( Mb, solid line). Inset, expanded plot of the 7110-7120 eV region of the spectra.



The preedge 1 s 3 d transitions of these three heme protein CO adducts are split into two peaks, A and B (Fig. 5, inset). This transition is generally electric dipole forbidden but quadrupole allowed and can gain intensity from 3 d-4 p mixing. The iron 3 d orbitals split into two degenerate e orbitals and three degenerate t orbitals in an perfect octahedral ligand field. For the low spin ferrous state, the three t orbitals are occupied by all six d electrons, leaving the e orbitals, d

High Spin Ferrous

The Fe-K edge spectra of deoxyferrous high spin chloroperoxidase and P-450-CAM are shown in Fig. 6 . It would be expected for the ferrous oxidation state that the edge position is less sensitive to the electron donating character of the axial ligand, as electron density at the iron is inherently higher. Ferrous-CO samples, on the other hand, contain an electron withdrawing CO ligand that decreases the electron density at the iron, leaving it more sensitive to the presence of the electron donating thiolate ligand. Although no crystal structure has been published for high spin deoxyferrous P-450-CAM, our previous EXAFS results for this state have clearly demonstrated that it has a sulfur donor axial ligand (23) . The shape and the edge position in the spectrum of deoxyferrous chloroperoxidase is very similar to that of deoxyferrous P-450-CAM (Fig. 6), suggesting that the electron density at the iron is similar for these two proteins. The only significant difference is the somewhat higher preedge intensity in the spectrum of deoxyferrous chloroperoxidase.


Figure 6: Comparison of the Fe-K x-ray absorption edge spectra of the deoxyferrous derivatives of cytochrome P-450-CAM ( P-450, dotted line) and chloroperoxidase ( CPO, solid line).



In summary, direct evidence has been obtained through the use of Fe-K x-ray absorption edge spectroscopy to show that a thiolate axial ligand is more electron releasing to the central heme iron than an imidazole axial ligand. In both the low spin six-coordinate ferric and ferrous states of cytochrome P-450, chloroperoxidase, or myoglobin, the absorption edge is shifted by about 1.0 eV to lower energy when comparing thiolate-ligated adducts with complexes of the same oxidation and spin state that lack a thiolate ligand. Because the edge also shifts to lower energy upon reduction of ferric iron to the ferrous state, the shift in the edge energy for thiolate-bound complexes provides a direct measure of the electron-releasing character of a thiolate axial ligand. These results therefore support the suggested role of the cysteinate proximal ligand of cytochrome P-450 as a strong internal electron donor to promote O-O bond cleavage in the putative ferric-peroxide intermediate to generate the proposed ferryl-oxo ``active oxygen'' state of the reaction cycle (1, 2, 3) .

  
Table: Relative edge position shift



FOOTNOTES

*
This research was supported by the National Science Foundation Grants CHE-9121576 (to K. O. H.) and DMB-8605876 (to J. H. D.) and National Institutes of Health Grants GM-07768 (to L. P. H.) and RR-01209 (to K. O. H.). X-ray absorption data were collected at the Stanford Synchrotron Radiation Laboratory, which is supported by the United States Department of Energy, Office of Basic Energy Sciences, Divisions of Chemical and Materials Sciences and Office of Health and Environmental Research and by the National Institutes of Health, Biomedical Research Technology Program, National Center for Research Resources. Additional data were collected at the National Synchrotron Light Source, Brookhaven National Laboratory, which is supported by the Department of Energy, Divisions of Materials and Chemical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: EXAFS, extended x-ray absorption fine structure; SSRL, Stanford Synchrotron Radiation Laboratory; NSLS, National Synchrotron Light Source.


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