Electron spin and electron nuclear double resonance of the [FeO2]− centre from irradiated oxyhemo- and oxymyoglobin

Kappl, Reinhard; Höhn-Berlage, M.; Hüttermann, Jürgen; Bartlett, Nicholas; Symons, Martyn C. R.

doi:10.1016/0167-4838(85)90218-3

Cited by 74 publications

(133 citation statements)

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“…there are 2 primary Mb02-species at 77 K which !lave different bonding geometry [5.6]. Also, for one of the species we have been able to observe a strong H-bond to the distal histidine (E7) by ENDOR-spectroscopy [5]. Further investigations will be necessary for the intermediate in P450,,,,,, in order to elucidate structural details of its stereochemistry and the mechanism of its conversion to hydroxylated product and ferric enzyme.…”

Section: Resultsmentioning

confidence: 97%

“…and oxyhemoglobin (HbO,) [5,6]. Furthermore, in X-irradiated samples of ferrous P450,,,, without oxygen or with carbon monoxide ligation, the characteristic rhom!…”

Section: Resultsmentioning

confidence: 99%

“…A way to overcome this obstacle is the application of Xirradiation-generated electrons in situ, at 77 K. We have shown previously that kinetically stabilized adduct complexes with heme-oxygen complexes as appropriate electron acceptors can be formed by irradiation of frozen aqueous solutions of oxymyo-and oxyhemoglobin [5,6]. Applying this technique to study the structural features in the initial product of the reduction of the ternary complex of cytochrome P450,,,,, we wish to prcsent the first EPR-results of the observation of a new intermediate.…”

Section: Introductionmentioning

confidence: 99%

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EPR‐spectroscopy of reduced oxyferrous‐P450_cam

et al. 1991

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Section: Resultsmentioning

confidence: 97%

“…and oxyhemoglobin (HbO,) [5,6]. Furthermore, in X-irradiated samples of ferrous P450,,,, without oxygen or with carbon monoxide ligation, the characteristic rhom!…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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EPR‐spectroscopy of reduced oxyferrous‐P450_cam

et al. 1991

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“…A difficulty in the case of the steroidogenic cytochromes P450 is the inherently high reactivity of the encountered peroxo-and hydroperoxo-intermediates, coupled with impressively efficient delivery of protons to the active site Fe-O-O fragment. This obstacle has made temporal isolation of these fleeting species especially challenging, requiring the application of cryoradiolysis, a technique applied successfully to many systems by Symons, Hoffman, and their coworkers (20)(21)(22). Here, this low-temperature method allows reduction of a stabilized ferrous dioxygen state while effectively restricting associated proton transfer, as we have shown for other P450 systems (21)(22)(23)(24).…”

Section: Significancementioning

confidence: 84%

Unveiling the crucial intermediates in androgen production

Mak

Gregory

Denisov

et al. 2015

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

189

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Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C 17 -C 20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C 20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon-carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.T he excessive production of androgen, which effectively fuels the progression of cancer, especially prostate cancer, was first treated by surgical methods (1), whereas more modern approaches are focused on the discovery and development of pharmaceuticals that can selectively inhibit androgen synthesis (2, 3). A member of the cytochrome P450 superfamily (4, 5), cytochrome P450 17A1 (CYP17A1), occupies a central role in the biosynthesis of steroid hormones in humans. As was first reported by Nakajin and Hall (6) and Nakajin et al. (7) for CYP17 from pig testis, this enzyme catalyzes two fundamentally different types of chemical transformations (5-11), with the first being the efficient hydroxylation of both of its primary substrates, pregnenolone (PREG) and progesterone (PROG), to 17-hydroxypregnenolone (17-OH) PREG and 17-hydroxypregnenolone, respectively (Fig. 1). Importantly, 17-OH PREG is further processed in a second step in which CYP17A1 now catalyzes, not another hydroxylation reaction, but a complex 17,20 carbon-carbon (C-C) bond cleavage (lyase) reaction. This step converts 17-OH PREG to dehydroepiandrosterone (DHEA), a process that represents a critical branch point in human steroidogenesis by providing the essential precursor to androgens and various corticosteroids (5-9). Although a similar C-C bond cleavage of 17-OH PROG is also mediated by CYP17A1, it is of less importance in humans because its efficiency is only about 2% of the efficiency of the physiologically important lyase reaction involving 17-OH PREG (10). Recognizing the intensive efforts currently underway to design and test substances that selectively inhibit CYP17A1 (2, 3), there is a pressing need to enhance our understanding of the relevant reaction mechanisms, a task that typically entails identification of key reaction intermediates. Dir...

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“…(9); (---) kinetic rate for 5-hydroxycamphor formation, k ) 0.0035 s -1 ; (---) kinetic rate for autooxidation of putidaredoxin, k ) 0.007 s -1 (Sligar et al, 1974); (s) kinetic rate for 1.0 equiv of oxidized putidaredoxin formed in 1 s and 1.1 equiv autooxidize at the same rate above, k ) 0.007 s -1 . Coon, 1981;Davydov et al, 1991;Kappl et al, 1985;Nyman & Debrunner, 1991;Tajima et al, 1993) and observed model compounds (Balch, 1992;Tajima et al, 1995;Yamaguchi et al, 1993). The spectral intermediate observed with D251N is distinct from these species in both its UV-visible spectra and EPR spectra (Balch, 1992;Blake & Coon, 1981;Davydov et al, 1991;Kappl et al, 1985;Nyman & Debrunner, 1991;Tajima et al, 1993Tajima et al, , 1995Yamaguchi et al, 1993), suggesting the rate-determining step is the first proton transfer (5A and B to 5C).…”

Section: Resultsmentioning

confidence: 99%

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

1997

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Cytochrome P450s are ubiquitous heme proteins responsible for various oxidative metabolic processes. The overall rate-determining step in the catalytic cycle of native cytochrome P450 cam is the reduction of the dioxygen complex, which has made detection of catalytic intermediates after this reduction impossible. However, for the site-specific mutant D251N cytochrome P450 cam (which affects proton transfer near the catalytic center), the overall rate-determining step occurs after the reduction of oxy-P450. As a consequence, we have observed in the UV-visible spectrum during catalytic turnover a new intermediate that is one electron reduced from oxy-P450 with an intact dioxygen bond.

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Electron spin and electron nuclear double resonance of the [FeO2]− centre from irradiated oxyhemo- and oxymyoglobin

Cited by 74 publications

References 12 publications

EPR‐spectroscopy of reduced oxyferrous‐P450_cam

EPR‐spectroscopy of reduced oxyferrous‐P450_cam

Unveiling the crucial intermediates in androgen production

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam

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Electron spin and electron nuclear double resonance of the [FeO2]− centre from irradiated oxyhemo- and oxymyoglobin

Cited by 74 publications

References 12 publications

EPR‐spectroscopy of reduced oxyferrous‐P450cam

EPR‐spectroscopy of reduced oxyferrous‐P450cam

Unveiling the crucial intermediates in androgen production

Reduced Oxy Intermediate Observed in D251N Cytochrome P450cam

Contact Info

Product

Resources

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EPR‐spectroscopy of reduced oxyferrous‐P450_cam

EPR‐spectroscopy of reduced oxyferrous‐P450_cam

Reduced Oxy Intermediate Observed in D251N Cytochrome P450_cam