Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN- complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.

Spectroscopic characterization of ferric soybean peroxidase with peroxides were studied to determine the ligand coordination and to characterize the structure of the heme active site and its intermediates (ferryl species). The lifetime, chemical reactivity and distinctive colors of the ferryl species (FeIV) formed during the oxidation of peroxidase (FeIII) by peroxides enabled structure, dynamics and reaction mechanisms to be studied. Resonance Raman spectroscopy was used as a means of characterizing the structure of the soybean peroxidase and its intermediates. Excitation in the Soret absorption band at 406.7nm with 2-5mW laser power was used for this study. Resonance Raman spectra in the 200 to 1700 cm-1 region were obtained for the soybean peroxidase. However, the focus of this study was on the vibrational region of the resonance Raman spectra from 900 to 500cm-1 where the FeIV=O stretching frequencies for heme compound II intermediates are expected. Several pH and pD (deuterium substitution) samples of the soybean peroxidase were analyzed using resonance Raman spectroscopy. The vibrational stretching frequencies of the ferryl peroxidases varied with varying pH/pD were observed within the 773–787cm-1 range. From the deuterium experiment, accompanied with changes in the vibrational frequencies of the iron-ligand, a 3cm-1 upshift and intense resonant enhancement of the peaks, we observed the ferryl nature of compound II intermediate for soybean peroxidase. Badger’s rule was used to estimate the bond distances that existed within Fe-O which offers additional insight into the structure of the ferryl species. The estimated bond distance for the soybean peroxidase was significantly less than Fe-O bond distances proposed by X-ray crystallographers for other peroxidases in the same family. Comparing the vibrational frequencies of the ferryl intermediates in soybean peroxidase to that in heme proteins portrayed the effect the protein environment has on the vibrational frequencies.

Soy peroxidase enzyme obtained from isoelectic precipitation procedures was added to all-purpose flour (APF) to assess its effects on the rheological properties and consumer acceptability of yeast bread. A pH 4.8 isoelectrically precipitated fraction from soybeans was used because it produced the most precipitate and had about the same peroxidase activity as the other fractions. Gluten strength was determined using a farinograph for seven treatment groups: control (all-purpose flour), bread flour, all-purpose flour + soy flour, bread flour + soy flour, all purpose flour + pH 4.8 precipitate, all-purpose flour + 15 mg soybean peroxidase, and all-purpose flour + 25 mg soybean peroxidase. Four types of yeast bread were baked for loaf volume determination, texture analysis, and consumer acceptability: a control loaf using only all-purpose flour, a reference loaf using all bread flour, a loaf with all purpose flour + whole soy flour, and a loaf with all-purpose flour + pH 4.8 soy precipitate. The APF+soy flour, bread flour, bread flour + soy flour, and the APF + pH 4.8 precipitate produced an improvement in the gluten strength and mixing tolerance compared to the control (p<0.05). However, the improvement by the addition of the pH 4.8 precipitate cannot be attributed to the peroxidase enzyme because peroxidase needs hydrogen peroxide as a substrate and no hydrogen peroxidase could be added to the farinogragh; therefore, it was concluded that the increase in gluten strength produced by the pH 4.8 soy precipitate was due to an unknown component present in the pH 4.8 fraction. No significant differences (p<0.05) were found in crumb or crust texture for any of the treatment groups. The addition of pH 4.8 precipitate to APF significantly decreased (p<0.05) loaf volume compared to bread made from bread flour. The results from sensory analysis showed there was no difference in preference for any of the breads. This study showed no conclusive evidence that peroxidase enzyme improved gluten strength or loaf volume of yeast bread, but further research is warranted. / Master of Science

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