Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

Abstract

Cryogenic radiolytic reduction is one of the most simple and convenient methods of generation and stabilization of reactive iron-oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxoferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases.

Unveiling the crucial intermediates in androgen production

“…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).…”

Nanodiscs in Membrane Biochemistry and Biophysics

“…339,340 In order to study other intermediates in the P450 reaction cycle, a frozen sample of oxygenated CYP17A1 or CYP19A1 in Nanodiscs was irradiated with γ-rays from a cobalt-60 source, generating the peroxo- or hydroperoxo-ferric complexes with good yield as discussed in the original publications and reviews. 278,384,385,393 Using this approach, unstable intermediates present in cytochrome P450 catalysis can be observed, which was not possible by other methods. 24,82,87–89,278–280,370,397 Note that these methods were initially developed for studies of soluble bacterial P450s, 278,355,375,384,398–402 and later applied to the membrane bound mammalian P450s using the Nanodisc platform.…”

“…278,384,385,393 Using this approach, unstable intermediates present in cytochrome P450 catalysis can be observed, which was not possible by other methods. 24,82,87–89,278–280,370,397 Note that these methods were initially developed for studies of soluble bacterial P450s, 278,355,375,384,398–402 and later applied to the membrane bound mammalian P450s using the Nanodisc platform. 24,87,88,280 …”

“…87 Here, the oxy-complexes of CYP17A1 in Nanodiscs with PREG and 17-hydroxy-PREG were frozen and reduced at 77 K by γ-irradiation from a 60 Co source to form peroxo-ferric complex stabilized in the frozen glycerol-buffer matrix, as reviewed. 278,375,384,385 Optical absorption and resonance Raman spectra measured at gradually increasing temperatures were used to identify a new previously unseen intermediate with a Soret band at ~407 nm and vibrational ν(Fe-O 2 ) and ν(O-O) modes at 579 cm −1 and at 791 cm −1 respectively with 17-hydroxy-PREG substrate. 87 No such intermediate was observed with PREG bound.…”

“…356,386,394 In P450 CYP3A4 and CYP17A1, the positions of the Soret band in the oxy-complex vary in wavelength, depending on the substrate bound. 278,279,339 This variation can be used in combination with the rR data to investigate the positioning and orientation of substrates with respect to the oxygen bound heme iron catalytic site. In analogy, additional information of this type can be obtained from spectral studies using carbon monoxide (CO) or cyanide (CN − ) in the presence and absence of substrates and inhibitors.…”