Thomas M. Makris

Cryotrapped reaction intermediates of cytochrome p450 studied by radiolytic reduction with phosphorus-32.

Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using 60Co γ-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe3+-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999)J. Am. Chem. Soc. 121, 10654–10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at ∼195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed.

Decarboxylation of Fatty Acids to Terminal Alkenes by Cytochrome P450 Compound I

OleT(JE), a cytochrome P450, catalyzes the conversion of fatty acids to terminal alkenes using hydrogen peroxide as a cosubstrate. Analytical studies with an eicosanoic acid substrate show that the enzyme predominantly generates nonadecene and that carbon dioxide is the one carbon coproduct of the reaction. The addition of hydrogen peroxide to a deuterated substrate-enzyme (E-S) complex results in the transient formation of an iron(IV) oxo π cation radical (Compound I) intermediate which is spectroscopically indistinguishable from those that perform oxygen insertion chemistries. A kinetic isotope effect for Compound I decay suggests that it abstracts a substrate hydrogen atom to initiate fatty acid decarboxylation. Together, these results indicate that the initial mechanism for alkene formation, which does not result from oxygen rebound, is similar to that widely suggested for P450 monooxygenation reactions.

Resonance Raman Characterization of the Peroxo and Hydroperoxo Intermediates in Cytochrome P450

Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450(cam) (CYP101) are reported. Owing to the fact that proton delivery to the active site is hindered in this mutant, the unprotonated peroxo-ferric intermediate is observed as the primary species after radiolytic reduction of the oxy-complex in frozen solutions at 77 K. In as much as previous EPR and ENDOR studies have shown that annealing of this species to approximately 180 K results in protonation of the distal oxygen atom to form the hydroperoxo intermediate, this system has been exploited to permit direct RR interrogation of the changes in the Fe-O and O-O bonds caused by the reduction and subsequent protonation. Our results show that the nu(O-O) mode decreases from a superoxo-like frequency near approximately 1130 cm(-1) to 792 cm(-1) upon reduction. The latter frequency, as well as its lack of sensitivity to H/D exchange, is consistent with heme-bound peroxide formulation. This species also exhibits a nu(Fe-O) mode, the 553 cm(-1) frequency of which is higher than that observed for the nonreduced oxy P450 precursor (537 cm(-1)), implying a strengthened Fe-O linkage upon reduction. Upon subsequent protonation, the resulting Fe-O-OH fragment exhibits a lowered nu(O-O) mode at 774 cm(-1), whereas the nu(Fe-O) increases to 564 cm(-1). Both modes exhibit a downshift upon H/D exchange, as expected for a hydroperoxo-ferric formulation. These experimental RR data are compared with those previously acquired for the wild-type protein, and the shifts observed upon reduction and subsequent protonation are discussed with reference to theoretical predictions.

Mechanistic enzymology of oxygen activation by the cytochromes P450.

The P450 cytochromes represent a universal class of heme-monooxygenases. The detailed mechanistic understanding of their oxidative prowess is a critical theme in the studies of metabolism of a wide range of organic compounds including xenobiotics. Integral to the O2 bond cleavage mechanism by P450 is the enzymes concerted use of protein and solvent-mediated proton transfer events to transform reduced dioxygen to a species capable of oxidative chemistry. To this end, a wide range of kinetic, structural, and mutagenesis data has been accrued. A critical role of conserved acid-alcohol residues in the P450 distal pocket, as well as stabilized waters, enables the enzyme to catalyze effective monooxygenation chemistry. In this review, we discuss the detailed mechanism of P450 dioxygen scission utilizing the CYP101 hydroxylation of camphor as a model system. The application of low-temperature radiolytic techniques has enabled a structural and spectroscopic analysis of the nature of critical intermediate states in the reaction.

Cryoradiolysis for the study of P450 reaction intermediates

Publisher Summary This chapter describes cryoradiolysis for the study of p450 reaction intermediates. Cryogenic entrapment is generally used in matrix isolation chemistry for structural and spectroscopic studies. A similar method has been adapted for biochemical needs in mechanistic studies of cytochrome P450cam and heme oxygenase. The method is based on preparing the precursor (oxyferrous heme complex) at ambient conditions, freezing it at 77 K, and irradiating it with high-energy photons (X-rays or gamma rays from a 6°C source). The chapter describes radiolysis. Radiolysis of the solvent water and glycerol provides electrons that reduce the oxyferrous heme complex and turn it into the active peroxo-heme or hydroperoxoheme complex. Working at cryogenic temperature in the frozen solution incorporates the advantages of radiolysis and avoids its drawbacks, the most important advantage being the multiple side reactions with different radical products of radiolysis. The majority of results on radiolysis of simple substances are obtained using gamma rays.

Structure of a Dinuclear Iron Cluster-Containing β-Hydroxylase Active in Antibiotic Biosynthesis

A family of dinuclear iron cluster-containing oxygenases that catalyze β-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Münck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 Å X-ray crystal structure of the archetypal enzyme from the family, CmlA, is reported. CmlA catalyzes β-hydroxylation of l-p-aminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of CmlA is unlike any previously reported, but the C-terminal domain has the αββα fold of the metallo-β-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of O2 activation.

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

Significance The biocatalytic production of hydrocarbons from abundant natural resources provides a means to expand the current fuel inventory. The recently identified cytochrome P450, OleT, synthesizes 1-alkenes from fatty acids through a carbon−carbon scission reaction that is highly atypical for the enzyme superfamily. Rapid kinetics studies reveal two critical reaction cycle intermediates that define the catalytic strategy used by OleT and highlight the functional versatility of P450 enzymes. OleT is a cytochrome P450 that catalyzes the hydrogen peroxide-dependent metabolism of Cn chain-length fatty acids to synthesize Cn-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe4+−oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe4+−OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT.

Expanding the substrate scope and reactivity of cytochrome P450 OleT.

The efficient hydrogen peroxide-dependent hydroxylation and epoxidation of hydrocarbons is catalysed by a P450 fatty acid decarboxylase (OleT) active-site variant. The introduction of an acidic functionality in the protein framework circumvents the necessity for a carboxylate that is typically provided by the substrate for efficient H2O2 heterolysis. Spectroscopic and turnover studies show that the mutation eliminates the binding and metabolism of prototypical fatty acid substrates, but permits the oxidation of a broad range of inert hydrocarbon substrates.

Mixed regiospecificity compromises alkene synthesis by a cytochrome P450 peroxygenase from Methylobacterium populi

Intensive interest has focused on enzymes that are capable of synthesizing hydrocarbons, alkenes and alkanes, for sustainable fuel production. A recently described cytochrome P450 (OleTJE) from the CYP152 family catalyzes an unusual carbon-carbon scission reaction, transforming Cn fatty acids to Cn-1 1-alkenes. Here, we show that a second CYP152, CYP-MP from Methylobacterium populi ATCC BAA 705, also catalyzes oxidative substrate decarboxylation. Alkene production is accompanied with the production of fatty alcohol products, underscoring the mechanistic similarity of the decarboxylation reaction with canonical P450 monooxygenation chemistry. The branchpoint of these two chemistries, and regiospecificity of oxidation products, is strongly chain length dependent, suggesting an importance of substrate coordination for regulating alkene production.

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry

OleT is a cytochrome P450 enzyme that catalyzes the removal of carbon dioxide from variable chain length fatty acids to form 1-alkenes. In this work, we examine the binding and metabolic profile of OleT with shorter chain length (n ≤ 12) fatty acids that can form liquid transportation fuels. Transient kinetics and product analyses confirm that OleT capably activates hydrogen peroxide with shorter substrates to form the high-valent intermediate Compound I and largely performs C-C bond scission. However, the enzyme also produces fatty alcohol side products using the high-valent iron oxo chemistry commonly associated with insertion of oxygen into hydrocarbons. When presented with a short chain fatty acid that can initiate the formation of Compound I, OleT oxidizes the diagnostic probe molecules norcarane and methylcyclopropane in a manner that is reminiscent of reactions of many CYP hydroxylases with radical clock substrates. These data are consistent with a decarboxylation mechanism in which Compound I abstracts a substrate hydrogen atom in the initial step. Positioning of the incipient substrate radical is a crucial element in controlling the efficiency of activated OH rebound.