Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.     

Sulfoxidation mechanisms catalyzed by cytochrome P450 and horseradish peroxidase models: spin selection induced by the ligand

The sulfoxidation of dimethyl sulfide (DMS), by two different heme-type enzyme models (without the protein), namely, horseradish peroxidase (HRP) and cytochrome P450 (P450), was studied using density functional theory. The models differ from each other by the axial ligand of the iron, which is imidazole in the case of HRP and thiolate in the case of P450. The computational results reveal a concerted oxygen atom transfer to sulfur, with spin-state selection dependent upon the identity of the proximal ligand. In the case of thiolate, the mechanism prefers the high-spin quartet pathway; whereas in the case of imidazole, the mechanism involves two-state reactivity (TSR), with competing quartet and doublet spin states. Furthermore, with thiolate the high-spin transition state, (4)TS(P450), has an upright conformation with a large Fe-O-S(DMS) angle of 147 degrees , whereas the low-spin species, (2)TS(P450), has a small angle and its Fe-O moiety makes an O-N(Por) bond with one of the nitrogen atoms of the porphine macrocycle. By contrast, when the proximal ligand is imidazole, both transition states possess a bent Fe-O bond and an O-N(Por) bond. These spin-state selection patterns obey simple orbital-selection rules, which are manifestations of the electronic nature of the ligand, i.e., the electron-releasing effect of the thiolate vis-a-vis the electron-withdrawal effect of imidazole. Other possible reactivity expressions of the spin-selection patterns are discussed [Dowers, T. S., Rock, D. A., Rock, D. A., Jones, J. P. (2004) J. Am. Chem. Soc. 126, 8868-8869]. Theory shows that intrinsically, HRP should be as reactive as P450 toward sulfoxidation.     

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.     

Elucidating the role of the proximal cysteine hydrogen-bonding network in ferric cytochrome P450cam and corresponding mutants using magnetic circular dichroism spectroscopy

Although extensive research has been performed on various cytochrome P450s, especially Cyt P450cam, there is much to be learned about the mechanism of how its functional unit, a heme b ligated by an axial cysteine, is finely tuned for catalysis by its second coordination sphere. Here we study how the hydrogen-bonding network affects the proximal cysteine and the Fe-S(Cys) bond in ferric Cyt P450cam. This is accomplished using low-temperature magnetic circular dichroism (MCD) spectroscopy on wild-type (wt) Cyt P450cam and on the mutants Q360P (pure ferric high-spin at low temperature) and L358P where the "Cys pocket" has been altered (by removing amino acids involved in the hydrogen-bonding network), and Y96W (pure ferric low-spin). The MCD spectrum of Q360P reveals fourteen electronic transitions between 15200 and 31050 cm(-1). Variable-temperature variable-field (VTVH) saturation curves were used to determine the polarizations of these electronic transitions with respect to in-plane (xy) and out-of-plane (z) polarization relative to the heme. The polarizations, oscillator strengths, and TD-DFT calculations were then used to assign the observed electronic transitions. In the lower energy region, prominent bands at 15909 and 16919 cm(-1) correspond to porphyrin (P) → Fe charge transfer (CT) transitions. The band at 17881 cm(-1) has distinct sulfur S(π) → Fe CT contributions. The Q band is observed as a pseudo A-term (derivative shape) at 18604 and 19539 cm(-1). In the case of the Soret band, the negative component of the expected pseudo A-term is split into two features due to mixing with another π → π* and potentially a P → Fe CT excited state. The resulting three features are observed at 23731, 24859, and 25618 cm(-1). Most importantly, the broad, prominent band at 28570 cm(-1) is assigned to the S(σ) → Fe CT transition, whose intensity is generated through a multitude of CT transitions with strong iron character. For wt, Q360P, and L358P, this band occurs at 28724, 28570, and 28620 cm(-1), respectively. The small shift of this feature upon altering the hydrogen bonds to the proximal cysteine indicates that the role of the Cys pocket is not primarily for electronic fine-tuning of the sulfur donor strength but is more for stabilizing the proximal thiolate against external reactants (NO, O(2), H(3)O(+)), and for properly positioning cysteine to coordinate to the iron center. This aspect is discussed in detail.     

Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

Trans-substitution of the proximal hydrogen bond in myoglobin: I. Structural consequences of hydrogen bond deletion

The structural role of a side-chain to side-chain protein hydrogen bond is examined using trans-substitution of the proximal histidine of myoglobin with methylimidazoles (Barrick, Biochemistry 1994;33:6546-6554). Modification of the chemical structure of exogenous ligands allows this hydrogen bond to be disrupted. Comparison of the crystal structures of H93G myoglobin complexed 4-methylimidazole (4meimd; methylation at carbon 4) and 1-methylimidazole (1meimd; methylation at the adjacent nitrogen, preventing hydrogen bonding between the imidazole ligand and the protein) shows that the polypeptide, heme, and methylimidazole orientations are the same within error. For 4meimd there appear to be major and minor conformations corresponding to different tautomeric states of the ligand. Conformational heterogeneity is also seen in the hyperfine-shifted region of the NMR spectrum of 4meimd complexed with high-spin H93G deoxyMb. The major conformation of the 4meimd ligand and the 1meimd ligand, as seen in the respective crystal structures, are quite similar except that the proximal ligand NH-to-Ser92-OH hydrogen bond is eliminated in the 1meimd complex, and instead the proximal ligand CH is adjacent to the Ser92-OH. Thus, this system provides a means to eliminate the Mb proximal hydrogen bond in a chemically and structurally conservative way.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character.

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved. There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein structure.     

Spectroscopic characterization and ligand-binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1.

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated 'narrow' (gx,y,z = 6.24, 5.42, 2.00) and 'broad' (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = -23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.     

Fe–O versus O–O bond cleavage in reactive iron peroxide intermediates of superoxide reductase

It is generally accepted that the catalytic cycles of superoxide reductases (SORs) and cytochromes P450 involve a ferric hydroperoxo intermediate at a mononuclear iron center with a coordination sphere consisting of four equatorial nitrogen ligands and one axial cysteine thiolate trans to the hydroperoxide. However, although SORs and P450s have similar intermediates, SORs selectively cleave the Fe–O bond and liberate peroxide, whereas P450s cleave the O–O bond to yield a high-valent iron center. This difference has attracted the interest of researchers, and is further explored here. Meta hybrid DFT (M06-2X) results for the reactivity of the putative peroxo/hydroperoxo reaction intermediates in the catalytic cycle of SORs were found to indicate a high-spin preference in all cases. An exploration of the energy profiles for Fe–O and O–O bond cleavage in all spin states in both ferric and ferrous models revealed that Fe–O bond cleavage always occurs more easily than O–O bond cleavage. While O–O bond cleavage appears to be thermodynamically and kinetically unfeasible in ferric hydrogen peroxide complexes, it could occur as a minor (significantly disfavored) side reaction in the interaction of ferrous SOR with hydrogen peroxide.     

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.     

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam.

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm-1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O-O bond.     

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.     

Density Functional Theory Calculations on Fe-O and O-O Cleavage of Ferric Hydroperoxide Species: Role of axial ligand and spin state

Density functional theory (DFT) calculations are performed on thiolate bound hydroperoxide complexes. O-O and Fe-O cleavage reaction coordinates, relevant to the active sites of cytochrome P450 and superoxide reductase enzymes, were investigated for both high and low spin states and for cis and trans orientations of the thiolate ligand with respect to the hydroperoxide ligand. The results indicate that the presence of a thiolate ligand produces significant elongation of the Fe-O bond and reduction of Fe-O vibrational frequency. While the fate of the O-O cleavage reaction is not significantly altered, the presence of a thiolate induces a heterolytic Fe-O cleavage irrespective of the spin state and orientation which is very different from results obtained with a trans ammine ligand.     

Comparative EPR study on high-spin ferric porphine complexes and cytochrome P-450 having rhombic character

Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved.There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein stucture.     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron paramagnetic resonance investigations of exogenous ligand complexes of low-spin ferric chloroperoxidase: further support for endogenous thiolate ligation to the heme iron.

Previous spectroscopic studies of chloroperoxidase have provided evidence for endogenous thiolate sulfur donor ligation to the central heme iron of the enzyme. This conclusion is further supported by recent DNA sequence data which revealed the existence of a third cysteine residue (in addition to a disulfide pair detected earlier) in the protein available for coordination to the heme iron. Thus, chloroperoxidase shares many spectroscopic properties with cytochrome P-450, the only other known thiolate-ligated heme protein. Surprisingly, a previous electron paramagnetic resonance (EPR) study of low-spin ferric chloroperoxidase-ligand complexes (Hollenberg, P.F., Hager, L.P., Blumberg, W.E. and Peisach, J. (1980) J. Biol. Chem. 255, 4801-4807) was unable to provide clear support for the presence of a thiolate ligand, although sulfur coordination was implicated. This was, in part, because an insufficient number of complexes was examined. In this work, we have significantly expanded upon the previous EPR study by using an extensive variety of over twenty exogenous ligands including carbon, nitrogen, oxygen, phosphorus and sulfur donors. Crystal field analysis, using the procedure of Blumberg and Peisach, of the present data in comparison with data for analogous complexes of cytochrome P-450-CAM, thiolate-ligated heme model systems, and myoglobin, is clearly indicative of endogenous thiolate ligation for chloroperoxidase. In addition, the UV-visible absorption and EPR spectral data suggest that a carboxylate ligand is a possible candidate for the endogenous sixth ligand to the heme iron that is responsible for the reversible conversion of ferric chloroperoxidase from high-spin to low-spin at low temperatures (less than 200 K).     

Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism.

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.     

Resonance Raman detection of the Fe-S bond in endothelial nitric oxide synthase

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.