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Electron-nuclear double resonance spectra of horseradish peroxidase Compound I exhibit resonances from 14N and beta-protons, and most probably alpha-protons as well. The measurements prove that the oxyferryl iron of this enzymic intermediate is spin-coupled to a porphyrin radical. Comparison of experimentally obtained average pi-electron spin densities with theoretical predictions for a 4-fold symmetric porphyrin pi-cation radical is supportive of a 2A2u assignment for the radical's electronic state.

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.

A simplified and streamlined purification scheme has been developed for the large scale isolation of a green hemoprotein from human erythrocytes. The isolation procedure involves hypotonic lysis, freezing of the hemolysate at -60 degrees, centrifugation, direct chromatography of the supernatant on DEAE-cellulose, and subsequent cation exchange, anion exchange, and gel filtration chromatography. Approximately 2 mumol of this anionic hemoprotein were isolated per liter of packed erythrocytes. The previous inability to separate the prosthetic group from the protein by conventional solvent extraction procedures was shown to be a consequence of the highly polar character of the heme and not due to covalent linkage between heme and protein. The polar nature and marked lability of the heme necessitated development of techniques for the extraction, purification, and derivatization of the prosthetic group. The heme was separated from the protein by membrane filtration in the presence of pyridine and alkali or by disc gel electrophoresis in the presence of cyanide. The heme was methylated with trimethyloxonium tetrafluoroborate and further derivatized. The heme derivatives were purified on columns of Sephadex LH-20 or alumina. Chromatography of the heme, heme methyl ester, acetylated heme ester, and the corresponding porphyrin derivatives suggests that the heme contains three carboxyl groups and one or more polar, acetylatable functional groups, probably hydroxyl groups. Spectral characterization of these compounds, as well as the derivatives resulting from reaction of the heme with NH2OH, NaHSO3, and Na2S2O4, show that the prosthetic group is a previously undescribed, formyl-containing heme that can be clearly distinguished from heme a, Spirographis heme, and all other naturally occurring prosthetic groups.

The crystal structures of acid metmyoglobin and deoxy cobalt(II)mesoporphyrin IX myoglobin were compared by a difference Fourier analysis at 2.5 A resolution. No large differences in protein conformation were observed. The greatest density of structural differences was found in the heme region. There was a loss of the histidine-bound sulfate ion and of the metal-bound water molecule, as well as a shift in the position of the prosthetic group with associated changes in the adjacent globin. The structural changes resulting from the substitution of ethyl for the vinyl side chains of the porphyrin were clearly observed. There was also a suggestion of a conformational change of the porphyrin ring. It was not clear whether there was any change of the metal position relative to the porphyrin plane or proximal histidine.

It is shown that the magnesium-protoporphyrin-6-ester pool of plants is made up of monovinyl and divinyl components which were separated by chromatography on thin layers of polyethylene. At 77K, in ether, monovinyl-Mg-protoporphyrin-6-ester exhibited fluorescence emission and excitation maxima at 589 and 417 nm, respectively. The divinyl-Mg-protoporphyrin-6-ester component exhibited red-shifted emission and excitation maxima at 591 and 424 nm respectively. Demetallation of the monovinyl and divinyl Mg-protoporphyrin ester components converted them into monovinyl and divinyl-protoporphyrin IX free bases. Catalytic Mg-mesoporphyrin ester and the demetallated free bases into mesoporphyrin ester, Monovinyl-Mg-porphyrin chemical species were also detected in the Mg-protoporphyrin IX and the Mg-protoporphyrin IX diester pools of plants. It is proposed that the Mg-protoporphyrin IX, Mg-protoporphyrin IX-6-ester, and Mg-protoporphyrin IX-diester pools of plants are heterogenous and are made up of monovinyl and divinyl chemical species.

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.

The prosthetic heme of cytochrome P-450 is converted during the metabolism of ethylene into an abnormal hepatic porphyrin. This porphyrin has been isolated, purified, and characterized by field desorption mass spectrometry and 360-MHz nuclear magnetic resonance spectroscopy. It has been unambiguously identified as one of the four possible isomers of N-(2-hydroxyethyl)protoporphyrin IX (isolated as the dimethyl ester). The isomer which is obtained bears the N-alkyl group on one of the two propionic acid-substituted pyrrole rings in the porphyrin. Strong support is provided by the structure of this porphyrin for our contention that the prosthetic heme of cytochrome P-450 is alkylated during attempted transfer of the catalytically-activated oxygen to the pi-bond of destructive unsaturated substrates.

In spite of numerous spectroscopic similarities between chloroperoxidase and cytochrome P-450 (P-450) which suggest endogenous cysteinate axial ligation in chloroperoxidase as has been established for P-450, assignment of the endogenous axial ligand of chloroperoxidase has remained controversial since no available free sulfhydryl groups have been detected in chemical studies of chloroperoxidase. To help clarify this problem, we have carried out extensive studies of thiol-binding properties of native ferric chloroperoxidase and have compared our new results with those previously obtained in our laboratory on thiol adducts of P-450. We have found that the ligation of exogenous thiols to the heme iron of chloroperoxidase generates hyperporphyrin (split Soret) spectra (lambda max = approximately 372 and approximately 455 nm), consistent with the formation of bisthiolate low-spin ferric heme adducts as has been established for P-450 and its heme models. However, in contrast to the results with P-450, thiols not only coordinate to the ferric heme iron of chloroperoxidase in the thiolate form in competition with cyanide but also bind, presumably in the thiol form, to a site in the heme vicinity other than the heme iron without competition with cyanide. The thiol acidity (pK alpha greater than 7) or medium pH (pH 3-7) have little effect on the spectral and equilibrium properties of the adducts. Thiol binding to the latter site, which is presumably the organic substrate-binding site, causes a red shift of the Soret peak (339 vector approximately 420 nm) of the high-spin ferric enzyme; the resulting thiol adducts are still predominantly high spin. Similar spectral changes are also observed upon binding of other neutral sulfur (sulfides and disulfide) and oxygen (alcohols and ketones) donor ligands to native ferric chloroperoxidase. In conclusion, the generation of hyperporphyrin spectra upon exogenous thiol binding to native ferric chloroperoxidase provides considerable support for the presence of an endogenous thiolate ligand to the heme of the enzyme in its ferric state.