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A simple technique is described that uses a continuous wave laser with electromechanical modulation to obtain time-resolved Raman spectra of transient species on the millisecond timescale. The time behavior of the vibrational bands of the intermediates involved in the proton pumping of bacteriorhodopsin is determined. From these results, along with resonance enhancement and power dependence studies, the bands that appear in the continuous wave Raman spectrum of bacteriorhodopsin can be assigned to three intermediates in the photochemical cycle of bacteriorhodopsin, bR570, bL550, and bM412. The Raman spectra of bR570 and bM412 are compared with published spectra of model Schiff bases of all-trans and 13-cis retinal.

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.

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 α-β (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm−1can 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 v4frequency (primarily Ca-N) exhibits a 20 cm−1downshift. The v2, v11, and v37vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10–20 cm−1upshifts. The v3, v10, and v28frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3and v10are small, 3–5 cm−1; however, the downshift for v28is 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 π-cation radical.

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.

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 are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus. The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms. This result is in accord with the crystal structure of bovine catalase (Fita, I., and Rossmann, M.G. (1985) J. Mol. Biol.185, 21–37). In contrast, the crystallographic study of catalase from the ascomycete Penicillium vitale (Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A.A., Grebenko, A. I., Borisov, V. V., Bartels, K. S., Fita, I., and Rossmann, M. G. (1986) J. Mol. Biol. 188, 49–61) showed electron density on the distal side of the heme which could imply the presence of a sixth ligand, possibly a water molecule. However, both of these crystallographic studies showed the proximal ligand in catalase to be a tyrosine. The present study confirms tyrosinate coordination in each of the three catalases from the appearance of selected resonance-enhanced tyrosine vibrational modes. The most characteristic band is the tyrosinate ring mode at ∼ 1612 cm−1which is maximally enhanced with 488.0 nm excitation. The appearance of tyrosinate modes at 1607 and 1245 cm−1in the resonance Raman spectra of M. luteuscyano catalase serves to identify tyrosine as an axial ligand in bacterial as well as eukaryotic catalases. Unlike non-heme iron tyrosinate proteins, whose resonance Raman spectra are dominated by several intense bands diagnostic of tyrosine ligation, the heme-linked tyrosine modes are not easily distinguished from the large number of porphyrin vibrations.