-Deuteriation of magnesium porphyrins

Kenner, G. W.; Smith, Kevin M.; Sutton, Michael J. D.

doi:10.1016/s0040-4039(01)95925-8

Cited by 25 publications

(14 citation statements)

References 11 publications

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“…The d12-protoheme was prepared according to the method originally reported by Goff and coworkers,20 while d4-protoheme was obtained using well know procedures employing CH 3 OD as the deuterium source21 and as used previously in our laboratory 7,8,27,28. The effective deuteration at all four methyl groups in d12- and all four methine protons in d4-protohemin was checked by comparison of the 1 H NMR spectrum of the bis-cyano derivative of the deuterated analogue and the natural abundance starting material, as reported earlier 9,29.…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

“…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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“…Deuterium substitution at the four methine carbons (d4‐protoheme) and synthesis of 1,3,5,8‐(C 2 H 3 ) 4 ‐protoheme (d12‐protoheme) were accomplished according to a previously published procedure 32–35. Syntheses of 1,3‐(C 2 H 3 ) 2 ‐protoheme (1,3‐d6‐protoheme) and 5,8‐(C 2 H 3 ) 2 ‐protoheme (5,8‐d6‐protoheme) was conducted with small modifications of the procedure used for deuterium substitution of the four methyl groups of protoheme 34.…”

Section: Methodsmentioning

confidence: 99%

“…Thin layer chromatograms,40 pyridine hemochromogen electronic absorption spectra,41 and proton NMR (300 mHz) spectra34, 42 were acquired to check the chemical and isotopic purity of the final samples. The 1 H NMR spectra of the methine‐exchanged sample revealed that the meso‐positions were deuterated to the extent of 95% with a little loss (less than 5%) of vinyl protons 32, 42. Corresponding spectra of the base‐exchanged d12‐protoheme samples showed that all methyl groups in the protoheme‐d12 were deuterated to the extent of 95%.…”

Section: Methodsmentioning

confidence: 99%

Effects of systematic peripheral group deuteration on the low‐frequency resonance Raman spectra of myoglobin derivatives

et al. 2004

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Resonance Raman spectra are reported for a series of systematically deuterated analogues of myoglobin in its deoxy state as well as for its CO and O(2) adducts. Specifically, the myoglobin samples studied are those that have been reconstituted with deuterated protoheme analogues. These include the methine deuterated, protoheme-d4; analogue bearing C(2)H(3) groups at the 1, 3, 5, and 8 positions, protoheme-d12; the species bearing C(2)H(3) groups at the 1 and 3 positions only, 1,3-protoheme-d6; and the species bearing C(2)H(3) groups at the 5 and 8 positions only, 5,8-protoheme-d6. While the results are generally consistent with previously reported data for synthetic metalloporphyrin models and previous studies of labeled heme proteins, the high-quality low-frequency RR data reported here reveal several important aspects of these low-frequency modes. Of special interest is the fact that, using the two d6-protoheme analogues, it is shown that certain modes are apparently localized on particular pyrrole rings, while others are localized on different rings; i.e., several of these low-frequency modes are localized on one side of the heme.

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“…The oxyprotohemin was transferred to a mixture of ethyl acetate (50 ml) and glacial acetic acid (1 ml) under argon, and the ethyl acetate layer was washed three times with water and evaporated to dryness under reduced pressure, and the residue was used immediately. mesoDeuterated a-oxyprotohemin was prepared similarly from iron ,-oxo meso-deuterated protoporphyrin IX dimethyl ester made according to the method of Kenner et al (22).…”

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confidence: 99%

On the mechanism of the chemical and enzymic oxygenations of alpha-oxyprotohemin IX to Fe.biliverdin IX alpha.

Sano¹,

Sano²,

Morishima³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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-Deuteriation of magnesium porphyrins

Cited by 25 publications

References 11 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

Effects of systematic peripheral group deuteration on the low‐frequency resonance Raman spectra of myoglobin derivatives

On the mechanism of the chemical and enzymic oxygenations of alpha-oxyprotohemin IX to Fe.biliverdin IX alpha.

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