Cryoradiolysis as a Method for Mechanistic Studies in Inorganic Biochemistry

Spectroscopic studies of the cytochrome P450 reaction mechanisms

“…Recently these cryoradiolyticaly generated intermediates have been characterized using EPR, UV-Vis and rR spectroscopies. [37,38,39,40,41] Compound I is also very unstable and only recently has been trapped in several P450 enzymes using peroxide shunt pathway with artificial oxidant. [20,29] Sometimes a product complex can be identified by EPR and ENDOR following annealing of cryoreduced oxy complexes in several cytochromes P450.…”

“…However, the interrogation of the subsequent highly oxidizing intermediates has been hampered by the rapid decay of these species for reactions conducted in solution. Fortunately, the obstacles to characterization of these key species have been overcome by application of the cryoradiolytic reduction, a technique initially pioneered by the Symons group [52,53,54] and by Davydov [55,56,57] and more recently refined and extended by Hoffman, Sligar and coworkers, [15,38,39,42,58] with the most recent advance being the application of rR detection by the Sligar and Kincaid groups. [41,59,60]…”

Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H-Bond Network in Oxidase Activity of an Engineered Myoglobin

“… 10 Although oxy-heme is an electronically closed-shell system with no EPR signal, EPR spectroscopy has been used to offer insight into the properties of oxy-heme, e.g., its interactions with nearby residues and its intermediates upon reduction, by a method called cryoreduction. 11 Briefly, the oxy-heme protein, trapped at 77 K, is exposed to γ-radiation, releasing “free” electrons into the sample matrix to efficiently reduce EPR-silent oxy-heme into EPR-active peroxo-heme. Because cryogenic trapping suppresses thermal relaxation and equilibration of the protein and its active site, EPR spectra of the resulting sample, maintained at 77 K, provide valuable structural information on the oxy-heme precursor ( Scheme 2 ).…”

“…Because cryogenic trapping suppresses thermal relaxation and equilibration of the protein and its active site, EPR spectra of the resulting sample, maintained at 77 K, provide valuable structural information on the oxy-heme precursor ( Scheme 2 ). 10a , 10f − 10l , 11 Furthermore, the peroxo-heme products are intermediates along the pathway of O 2 reduction, and subsequent decay, achieved by stepwise warming of the samples, followed by rapid recooling to 77 K after each step to trap intermediates-a procedure called annealing, provides further insight into the reaction ( Scheme 1 ). 10a , 11 , 12 As a result, EPR spectroscopy in combination with cryoreduction and annealing has been used over the past two decades to probe the structure and interactions of O 2 in many heme proteins, including myoglobins and monooxygenases.…”

“…10a , 11 , 12 As a result, EPR spectroscopy in combination with cryoreduction and annealing has been used over the past two decades to probe the structure and interactions of O 2 in many heme proteins, including myoglobins and monooxygenases. 10a , 10f − 10q , 11 , 12 Because WTMb, the scaffold for F33Y-Cu B Mb, was among the first and most studied proteins by the above method, the abundance of results facilitates understanding of the latter by direct comparison. In this study, F33Y-Cu B Mb contained heme but no metal ion in the Cu B site, because a previous report showed that this protein exhibits high oxidase activity without a metal ion at Cu B , 9d mimicking cyt bd -oxygen reductases, which lack this site.…”