Driving Force for Oxygen‐Atom Transfer by Heme‐Thiolate Enzymes

Abstract

The heme-thiolate peroxygenase from Agrocybe aegerita (AaeAPO, EC 1.11.2.1) is a versatile biocatalyst and cytochrome P450 analogue which catalyzes a variety of oxygenation reactions with high efficiency and selectivity.[1] Our recent kinetic characterization of AaeAPO-catalyzed reactions has shown that AaeAPO compound I is an oxo-Fe IV porphyrin radical cation.[2] The reactivity of AaeAPO-I toward a panel of substrates showed very fast C À H hydroxylation rates, similar to those of cytochrome P450 (CYP119-I), [3] and much faster than chloroperoxidase compound I (CPO-I).[4] Mechanistic probes have revealed a large hydrogen isotope effect for aliphatic CÀH hydroxylation and rearranged products from the hydroxylation of norcarane. [1b] There is, however, very little information available regarding the thermodynamic properties of such highly reactive oxo-Fe species for any heme-thiolate proteins.For hydrogen abstraction reactions, the redox potential of the oxidant is correlated with the rates of CÀH activation. [5] Yet these values are often not accessible, especially for highly reactive oxidants. We have developed a method to measure redox potentials for oxometalloporphyrin model compounds and it takes advantage of the rapid, reversible oxygen-atom transfer between oxo-metal complexes and halide ions.[6] By using rapid-mixing stopped-flow spectroscopy, rate constants of both forward and reverse reactions are measured. Thus, the driving force of the unknown oxo-transfer redox couple [8]Herein, we describe measurements of the driving force for oxygen-atom transfer by the heme-thiolate proteins AaeAPO and CPO. We have found that oxo-transfer between AaeAPO-I and chloride or bromide ions is fast and reversible (Scheme 1). The redox potential of the couple AaeAPO-I/ ferric-AaeAPO has been obtained over a wide pH range from the rate constants of the forward and reverse reactions. Thus, the highly reactive AaeAPO-I can be placed on an absolute energy scale and compared with those of CPO and horseradish peroxidase (HRP) for the first time.AaeAPO-I was generated by the stoichiometric reaction of Fe III /AaeAPO with HOCl or HOBr and characterized by rapid-mixing, stopped-flow spectroscopy. The UV/Vis spectral features of AaeAPO-I generated with these hypohalous acids (Figure 1) are the same as those we recently reported for peroxyacid oxidations.[2] The Soret band of the ferric enzyme at l = 417 nm diminished over the first 50 ms after mixing while new absorbances, characteristic of the formation of an oxo-Fe IV porphyrin radical cation, appeared at l = 361 and 694 nm. AaeAPO-I subsequently decayed in a second, slower phase. Singular value decomposition (SVD) analysis of these transient spectra indicated that only two species were present in significant amounts during this transformation. The

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

“…We assign this spectrum to APO-II based on the characteristic split Soret band (370 nm and 428 nm) and two Q bands centered at 535 nm and 567 nm, slightly blue shifted from those of the ferric state (18). APO-I is the dominant intermediate by reacting ferric APO with an oxidant only, such as mCPBA (29,30). However, with the presence of nitroxyl radical, the accumulation of APO-I was not apparent.…”

“…This intermediate was shown to be highly competent for the hydroxylation of even strong C−H bonds. Further, APO-I reacts with chloride and bromide ions rapidly and reversibly, allowing a direct determination of the thermodynamics of oxygen transfer by APO-I (E' = 1.2 V) (30). Here, we report a previously unidentified method of generating the elusive ferryl state, APO-II, directly from APO-I over a wide range of pH.…”

Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

“…S8), would lead to generation of a carbocation poised for C−Cα cleavage to liberate CO 2 . Although the intrinsic reactivity of Ole-II is most likely undervalued here, largely due to limitations that stem from its method of preparation, the estimated reduction potential (51) and oxidative prowess of APO-II (46) serve as a useful guide. As olefin and alcohol products result from the metabolism of chemically similar substrates, both rigid and precise positioning of a carbon-centered radical would seem necessary to inhibit oxygen rebound.…”

Activation of Molecular Oxygen in Cytochromes P450

“…Despite the great variety of chemical transformations catalyzed by cytochromes P450, the vast majority of them are undoubtedly driven by Compound I This ferryl-oxo intermediate with a π-cation radical delocalized on the porphyrin is a very reactive species All attempts to observe this species in a P450 system using atmospheric dioxygen have so far failed However, important spectroscopic characterization and reactivity measurements have been obtained by using the [227][228][229][230] or by analogy to other closely related thiolate-ligated heme enzymes such as CPO [231][232][233] and peroxygenases [234,235], for which Comopund I is much more stable This situation changed with the work of Rittle and Green who achieved a breakthrough on the peroxide pathway by radically improving the purification protocol for thermostable CYP119 from the extremophile archae Sulfolobus acidocaldarius [236][237][238] Careful multistep removal of endogenous substrate analogs from the purified, heterologously expressed protein, which hampered earlier studies [222], allowed them to dramatically increase the yield of Compound I in a stopped-flow reaction with m-CPBA, reach-ing a conversion of greater than 75 % [236] This made possible high-precision UV-vis spectra to quantitate the reaction kinetics, which in turn provided the necessary information for the preparation of highly concentrated samples for EPR and Mössbauer spectroscopy The UV-vis spectra of Compound I confirmed the main features of the ferryl-oxo π-cation radical known from the earlier experiments: a broad Soret band at 367 nm and a pronounced charge-transfer band at 690 nm The EPR spectrum of CYP119 Compound I [236] had a different shape as compared to that previously reported for CPO, another thiolate-ligated heme protein [239] Fitting of both spectra to the S = 1 Fe(IV)-oxo unit coupled with S = 1/2 porphyrin radical resulted in a higher ratio of the exchange coupling ( J) to zero-field splitting ( D) for CYP119 ( J/D = 1.3) than in CPO ( J/D = 1.02) [236] The higher J value in CYP119 was tentatively attributed to either a higher spin density on the thiolate sulfur atom or a shortened Fe-S bond The Mössbauer parameters measured for the CYP119 Compound I were more similar to those of CPO [239], with the isomer shift δ = 0.11 mm/s (0.13 mm/s for CPO) and quadrupole splitting ΔE Q = 0.96 mm/s (0.90 mm/s for CPO) These parameters also correspond to the ferryl-oxo S = 1 unit exchange coupled to the porphyrin radical (S = 1/2).…”