Electron paramagnetic resonance and spectrophotometric studies of the peroxide compounds of manganese-substituted horseradish peroxidase, cytochrome-c peroxidase and manganese-porphyrin model complexes

Peroxide compounds of manganese protoporphyrin IX and its complexes with apo-horseradish peroxidase and apocytochrome-c peroxidase were characterized by electronic absorption and electron paramagnetic resonance spectroscopies. An intermediate formed upon titration of Mn(III)-horseradish peroxidase with hydrogen peroxide exhibited a new electron paramagnetic resonance absorption at g = 5.23 with a definite six-lined 55Mn hyperfine (AMn = 8.2 mT). Neither a porphyrin pi-cation radical nor any other radical in the apoprotein moiety could be observed. The reduced form of Mn-horseradish peroxidase, Mn(II)-horseradish peroxidase, reacted with a stoichiometric amount of hydrogen peroxide to form a peroxide compound whose electronic absorption spectrum was identical with that formed from Mn(III)-horseradish peroxidase. The electronic state of the peroxide compound of manganese horseradish peroxidase was thus concluded to be Mn(IV), S = 3/2. Mn(III)-cytochrome-c peroxidase reacted with stoichiometry quantities of hydrogen peroxide to form a catalytically active intermediate. The electronic absorption spectrum was very similar to that of a higher oxidation state of manganese porphyrin, Mn(V). Since the peroxide compound of manganese cytochrome-c peroxidase retained two oxidizing equivalents per mol of the enzyme (Yonetani, T. and Asakura, T. (1969) J. Biol. Chem. 244, 4580-4588), this peroxide compound might contain an Mn(V) center.     

Resonance Raman spectroscopy of horseradish peroxidase derivatives and intermediates with excitation in the near ultraviolet

Resonance Raman enhancement of derivatives and intermediates of horseradish peroxidase in the near ultraviolet (N-band excitation) results in intensity and enhancement patterns that are different from those normally observed within the porphyrin Soret (B-band) and alpha-beta (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm-1 can be assigned to porphyrin vibrational modes that are characteristically shifted in frequency due to removal of an electron from the porphyrin ring. The resonance Raman frequency shifts follow normal mode compositions. Relative to resonance Raman spectra of compound II, the v4 frequency (primarily Ca-N) exhibits a 20 cm-1 downshift. The v2, v11, and v37 vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10-20 cm-1 upshifts. The v3, v10, and v28 frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3 and v10 are small, 3-5 cm-1; however, the downshift for v28 is 14 cm-1. These frequency shifts are consistent with those of previously published resonance Raman studies of model compounds. In contrast to reports from other laboratories, the data presented here for horseradish peroxidase compound I can be attributed unambiguously to resonance Raman scattering from a porphyrin pi-cation radical.     

The porphyrin-sensitized photooxidation of horseradish apoperoxidase.

Horseradish apoperoxidase (apoHRP) was reconstituted with various porphyrin derivatives, e.g., ferric, cupric, manganese, and zinc protoporphyrin IX, metal-free protoporphyrin IX, hematoporphyrin IX and deuteroporphyrin IX. The visible absorption spectra of these porphyrin-apoHRP complexes were examined. The time required for maximum development of the new Soret peak after reconstitution was used to measure the rate of porphyrin-apoHRP reconstitution. All of the four metal-protoporphyrins reconstituted with apoHRP at the same rate as metal-free protoporphyrin IX, whereas, for the metal-free porphyrins, the rates of reconstitution were in the order of deuteroporphyrin IX > hematoporphyrin IX > protoporphyrin IX. The porphyrins on the reconstituted porphyrin-apoHRP complexes were used as localized photosensitizers for photodynamic studies. No amino acid residues were oxidized on illumination of the ferric, cupric and manganese protoporphyrin IX-apoHRP complexes due to the paramagnetic properties of these metal ions. With diamagnetic zinc ion, two histidine and one methionine residues were oxidized which was the same as in the protoporphyrin IX- and hematoporphyrin IX-apoHRP complexes. However, only one histidine was destroyed on illumination of the deuteroporphyrin IX-apoHRP complex. The results confirmed the resistance of horseradish peroxidase to photodynamic action and suggested the involvement of at least one histidine residue in the heme environment of horseradish peroxidase.     

Observation of the FeIV=O stretching vibration of ferryl myoglobin by resonance Raman spectroscopy

We have directly observed the oxyferryl group of ferryl myoglobin by resonance Raman spectroscopy. The FeIV = O stretching vibration is observed at 797 cm-1 and confirmed by an 18O-induced isotopic shift to 771 cm-1. The porphyrin center-to-nitrogen distance of ferryl myoglobin is significantly less than that previously observed for horseradish peroxidase compound II, which also contains an FeIV = O heme. The FeIII-CN- stretch of myoglobin (FeIII) cyanide is observed at 454 cm-1, which shifts to 449 cm-1 upon substitution with [13C]cyanide.     

Oxygen exchange between the Fe(IV) = O heme and bulk water for the A2 isozyme of horseradish peroxidase

Resonance Raman spectra were observed for compound II of horseradish peroxidase A2, and the Fe(IV) = O stretching Raman line was identified at 775 cm-1. This Raman line shifted to 741 cm-1 upon a change of solvent from H2(16)O to H2(18)O, indicating occurrence of the oxygen exchange between the Fe(IV) = O heme and bulk water. The oxygen exchange took place only at the acidic side of the heme-linked ionization with pKa = 6.9.     

Light absorption, electron paramagnetic resonance and resonance Raman characteristics of nitridochromium(V) protoporphyrin-IX and its reconstituted hemoproteins.

A surprisingly stable complex of the photolyzed product of azidochromium(III)protoporphyrin-IX was prepared and examined by light absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopies. The characteristic EPR spectrum for this complex was consistent with a nitridochromium(V)-porphyrin complex which was two oxidation equivalents above the resting Cr(III) complex. The Cr(V)-N stretching mode was observed at 1010 cm-1 by resonance Raman spectroscopy. A simple diatomic harmonic oscillation model gave a force constant of 6.7 mdyn/A for the Cr(V)-N bond, in the region characteristic for the metal-nitrogen triple bond. Nitridochromium(V) protoporphyrin-IX reconstituted myoglobin and cytochrome c peroxidase were prepared for the first time. The nitridochromium(V)-porphyrins in these apo-proteins were unstable in contrast with the protein-free chromium(V)porphyrin. Upon irradiation of the azide complexes of the chromium(III) protoporphyrin-IX reconstituted myoglobin and cytochrome c peroxidase with ultraviolet light aerobically at room temperature, the characteristic optical and EPR spectra for nitridochromium(V) derivatives were observed. The optical spectra of these photo-induced products were different from those of the nitridochromium(V) protoporphyrin-IX reconstituted hemoproteins. The electrochemical structures of the unusual metalloporphyrin seemed to be modulated by the heme surrounding amino acid residues.     

Copper(II) protoporphyrin IX as a reporter group for the heme environment in myoglobin.

Copper(II) protoporphyrin IX has been introduced into apomyoglobin, and its utility as a reporter group of the heme environment has been examined. The Soret and visible absorption bands and electron spin resonance spectrum show that the Cu(II) is five coordinate, probably through coordination to the F-8 proximal histidine. The resonance Raman spectrum does not indicate any appreciable distortion from the solution conformation of copper(II) protoporphyrin IX dimethyl ester in CS2. The ultraviolet circular dichroism shows no alteration of the helical content of the globin from that of metmyoglobin. The circular dichroism of the porphyrin transitions suggests that the packing of the amino acid side chains around the porphyrin is different than that in the native metmyoglobin.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

Resonance Raman evidence for an unusually strong exogenous ligand-metal bond in a monomeric nitrosyl manganese hemoglobin

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope-labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn11-NO stretching model at 628 cm-1, the Mn11-N-O bending mode at 574 cm-1 and the N-O stretching mode at 1735 cm-1. The results suggest a strong Mn11-NO bond and a weak N-O bond. The vinyl group substitution does not influence the nu (Mn11-NO), delta (Mn11-N-O) and nu (N-O) vibrations. The Mn11-NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N-O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an Mn111. NO complex which undergoes a slow but complete autoreduction resulting in the Mn11.NO species. In manganese meso-IX CTT IV, the Mn111. NO Mn11. NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm-1, but shifts the frequencies at 1504 and 1633 cm-1 (in Mn111.NO) to 1497 and 1630 cm-1 (in Mn11. NO), respectively. The unshifted marker line at 1378 cm-1 reflects the fact that the pi electron densities of the porphyrin ring are the same in the two complexes.     

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.     

Observation of the FeIV=O stretching Raman band for a thiolate-ligated heme protein. Compound I of chloroperoxidase.

The FeIV=O stretching vibration has never been identified for a cysteine-coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed-flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm-1, which shifted to 756 cm-1 with the 18O derivative, but the v4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin pi cation radical. The second intermediate gave an intense v4 band at 1,372 cm-1 but no oxygen isotope-sensitive Raman band, suggesting oxygen exchange with bulk water.     

Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization

Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme-linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron.     

Metal-ligand vibrations of cyanoferric myeloperoxidase and cyanoferric horseradish peroxidase: evidence for a constrained heme pocket in myeloperoxidase

The low-frequency FeCN vibrations of cyanoferric myeloperoxidase (MPO) and horseradish peroxidase (HRP) have been measured by resonance Raman spectroscopy. The ordering of the frequencies of the predominantly FeC stretching and FeCN bending normal vibrational modes in the two peroxidases differs. These normal mode vibrations are identified by their wavenumber shifts upon isotopic substitution of the cyanide ligand. For MPO, the stretching mode nu 1 (361 cm-1) occurs at a lower frequency than the bending mode delta 2 (454 cm-1). For HRP, the order is reversed as nu 1 (456 cm-1) is at a higher frequency than delta 2 (404 cm-1). Normal coordinate analyses and model complexes have been used to address the origin of this behavior. The nu 1 stretching frequencies in cyanide complexes of iron porphyrin and iron chlorin model compounds are similar to one another and to that of HRP. Thus, the inverted order and altered frequencies of the nu 1 and delta 2 vibrations in MPO, relative to those in HRP and the model compounds, are not inherent to the proposed iron chlorin prosthetic group in MPO but, rather, are attributed to distinct distal environmental effects in the MPO active site. The normal coordinate analyses for MPO and HRP showed that the nu 1 and delta 2 vibrational frequencies are not pure; the potential energy distributions for these modes respond not only to the geometry but also to the force constants of the nu(FeC) and delta(FeCN) internal coordinates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe(IV)=O stretching vibration

Fe(IV)=O resonance Raman stretching vibrations were recently identified by this laboratory for horseradish peroxidase compound II and ferryl myoglobin. In the present report it is shown that Fe(IV)=O stretching frequency for horseradish peroxidase compound II will switch between two values depending on pH, with pKa values corresponding to the previously reported compound II heme-linked ionizations of pKa = 6.9 for isoenzyme A-2 and pKa = 8.5 for isoenzyme C. Similar pH-dependent shifts of the Fe(IV)=O frequency of ferryl myoglobin were not detected above pH 6. The Fe(IV)=O stretching frequencies of compound II of the horseradish peroxidase isoenzymes at pH values above the transition points were at a high value approaching the Fe(IV)=O stretching frequency of ferryl myoglobin. Below the transition points the horseradish peroxidase frequencies were found to be 10 cm-1 lower. Frequencies of the Fe(IV)=O stretching vibrations of horseradish peroxidase compound II for one set of isoenzymes were found to be sensitive to deuterium exchange below the transition point but not above. These results were interpreted to be indicative of an alkaline deprotonation of a distal amino acid group, probably histidine, which is hydrogen bonded to the oxyferryl group below the transition point. Deprotonation of this group at pH values above the pKa disrupts hydrogen bonding, raising the Fe(IV)=O stretching frequency, and is proposed to account for the lowering of compound II reactivity at alkaline pH. The high value of the Fe(IV)=O vibration of compound II above the transition point appears to be identical in frequency to what is believed to be the Fe(IV)=O vibration of compound X.     

Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II

Resonance Raman spectroscopy has been used to investigate the structure and environment of the heme group in bovine liver catalase compound II. Both Soret- and Q-band excitation have been employed to observe and assign the skeletal stretching frequencies of the porphyrin ring. The oxidation state marker band v4 increases in frequency from 1373 cm-1 in ferricatalase to 1375 cm-1 in compound II, consistent with oxidation of the iron atom to the Fe(IV) state. Oxidation of five-coordinate, high-spin ferricatalase to compound II is accompanied by a marked increase of the porphyrin core marker frequencies that is consistent with a six-coordinate low-spin state with a contracted core. An Fe(IV) = O stretching band is observed at 775 cm-1 for compound II at neutral pH, indicating that there is an oxo ligand at the sixth site. At alkaline pH, the Fe(IV) = O stretching band shifts to 786 cm-1 in response to a heme-linked ionization that is attributed to the distal His-74 residue. Experiments carried out in H218O show that the oxo ligand of compound II exchanges with bulk water at neutral pH, but not at alkaline pH. This is essentially the same behavior exhibited by horseradish peroxidase compound II and the exchange reaction at neutral pH for both enzymes is attributed to acid/base catalysis by a distal His residue that is believed to be hydrogen-bonded to the oxo ligand. Thus, the structure and environment of the heme group of the compound II species of catalase and horseradish peroxidase are very similar. This indicates that the marked differences in their reactivities as oxidants are probably due to the manner in which the protein controls access of substrates to the heme group.     

The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.     

Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.     

Nickel(II) chelatase variants directly evolved from murine ferrochelatase: porphyrin distortion and kinetic mechanism

The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group

The green heme protein sulfmyoglobin (SMb) has been suggested to contain a sulfur-modified iron chlorin prosthetic group. To evaluate this hypothesis, we have obtained high-frequency (greater than 1000 cm-1) resonance Raman spectra of both oxidized and reduced SMb with 457.9-, 488.0-, 514.5-, 568.2-, and 647.1-nm excitation. The SMb spectra are compared to those of native met- and deoxymyoglobin (Mb). Vibrational frequencies for SMb are generally similar to those of Mb, suggesting a high-spin state for both the Fe(III) and Fe(II) SMb species, as is typical of native Mb. However, major differences between SMb and Mb occur both for patterns of relative spectral intensities and for depolarization ratios. In particular, all B1g-depolarized porphyrin modes in the Mb spectra have become polarized, totally symmetric vibrational modes in the SMb spectra. These contrasts reflect a dramatic lowering of the effective symmetry for the SMb prosthetic group. Several new bands are observed in SMb spectra that are not present in spectra of either native Mb or iron protoporphyrin IX complexes. The observation of additional polarized bands flanking the oxidation state marker, V4, is of particular interest. In a parallel study, we compared the resonance Raman spectral properties of iron protoporphyrin IX-derived chlorins and metallo-octaethylchlorins with those of the analogous porphyrins: the chlorin spectra exhibited altered intensity patterns, an increased number of totally symmetric (polarized) vibrational bands, and several new vibrational bands, including one or two in the region of the oxidation state marker, V4. Thus, the resonance Raman spectral characteristics of SMb and metallo-chlorins are complementary and strongly support a chlorin prosthetic group for SMb. Furthermore, they establish testable criteria for investigating the prosthetic group structures of other green heme proteins by resonance Raman spectroscopy.