Rapid base-catalyzed deuterium exchange at the ring-adjacent methyl and methylene positions of octaalkyl and natural-derivative porphyrins and metalloporphyrins

Godziela, Gregory M.; Kramer, Susan K.; Goff, Harold M.

doi:10.1021/ic00243a044

Cited by 18 publications

(22 citation statements)

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“…A measured purity index, R Z value,19 of 1.6 was indicative of high purity enzyme. The d12-protoheme was prepared by base catalyzed exchange of the 1,3,5,8-methyl groups (Figure 1) of natural protoheme in perdeuterated dimethyl sulfoxide by methods described previously9,20 and d4-protoheme by using procedures published previously 8,21…”

Section: Methodsmentioning

confidence: 99%

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

et al. 2008

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Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD3 groups at the 1,3,5 and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm−1. This substrate-activated mode had been previously assigned as a second “propionate bending” mode (Chen, Z.; Ost, T. W. B.; Schelvis, J. P. M. Biochemistry 2004, 43, 1798−1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm−1. In the present work, this newly activated mode is observed to shift by 8 cm−1 to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency “propionate bending” mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng, T. J.; Proniewicz, L. M.; Kincaid, J. R.; Yeom, H.; Macdonald, I. D. G.; Sligar, S. G. Biochemistry 1999 38, 13699−13706) that substrate binding activates several heme out-of-plane modes, one of which ( γ6) is accidentally degenerate with the 367 cm−1 propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle ν10 mode, has now allowed a definitive identification of the vinyl C=C stretching modes.

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Section: Methodsmentioning

confidence: 99%

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

et al. 2008

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“…A low temperature stirred cell (36) was employed for the measurement of Compound II species, while a conventional spinning cell was used for the resting state samples. Methyl group deuteration to give [ 2 H 12 ]mesohemin was carried out by the procedure described by Goff and co-workers (37). A sample of H 2 18 O 2 was prepared by a previously described procedure (38,39).…”

Section: Methodsmentioning

confidence: 99%

Resonance Raman Spectra of Native and Mesoheme-reconstituted Horseradish Peroxidase and Their Catalytic Intermediates

Kincaid

Zheng

Al-Mustafa

et al. 1996

Journal of Biological Chemistry

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Resonance Raman studies of native and mesohemereconstituted horseradish peroxidase and their catalytic intermediates, known as Compounds I and II, have been conducted using both near UV (ϳ350 nm) and visible (406.7 nm) excitation. Careful power studies indicate that the authentic Compound I spectra are obtainable using near UV excitation, but that use of visible excitation results in contamination of the Compound I spectrum with the spectrum of a Compound II-like photoproduct. Using H 2 18 O 2 , the (Fe‫؍‬O) stretching modes for both systems are unambiguously identified, for the first time, at ϳ790 cm ؊1 . The authentic Compound I spectra are indicative of an 2 A 1u -like ground state for both the native and the mesoheme-reconstituted proteins. Finally, the possible biological implications of such information are briefly discussed.

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“…30,42 Thin layer chromatography, pyridine hemochromogen electronic absorption spectra, and proton NMR were done to check purity of the final samples of labeled protohemins. 39,[43][44][45] The 1 H NMR spectra (300 Mz) in DMSO-d 6 /2 M KCN in D 2 O (1:6) revealed that the methyl groups in d12-protoheme were deuterated to the extent of >95% with little loss (<5%) of the vinyl or propionic protons, [38][39][40]43 while, in the case of 1,3-d6-protoheme, the 1 and 3 methyl groups were deuterated to the extent of 95%, while the 5 and 8 methyl groups remained essentially protonated (90-95%).…”

Section: Synthesis Of Selectively Deuterated Protohemesmentioning

confidence: 99%

Low frequency resonance Raman spectra of isolated α and β subunits of hemoglobin and their deuterated analogues

et al. 2006

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In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm(-1), are reported for the deoxy and ligated (CO and O2) forms of the isolated alpha and beta subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3-protoheme (d12- protoheme), the 1,3-C2H3-protoheme (1,3-d6-protoheme), the 5,8-C2H3-protoheme (5,8-d6-protoheme), and the meso-C2H4-protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm(-1), revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6- and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups.

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Rapid base-catalyzed deuterium exchange at the ring-adjacent methyl and methylene positions of octaalkyl and natural-derivative porphyrins and metalloporphyrins

Cited by 18 publications

References 0 publications

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Resonance Raman Spectra of Native and Mesoheme-reconstituted Horseradish Peroxidase and Their Catalytic Intermediates

Low frequency resonance Raman spectra of isolated α and β subunits of hemoglobin and their deuterated analogues

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