Biochemical and Biophysical Research Communications
ReviewThirty years of microbial P450 monooxygenase research: Peroxo-heme intermediates—The central bus station in heme oxygenase catalysis
Section snippets
A word about nomenclature for the uninitiated
By the early 1970s, several P450 monooxygenases had been identified. These included the membrane-bound human, rat, and rabbit enzymes involved in drug and steroid metabolism as well as a single bacterial P450 heme system. It was widely thought that there were but a couple dozen P450s, and they were either labeled by their presumed substrate (e.g., P450cam for camphor), by electrophoretic mobility (e.g., LM-2 for liver microsomal fraction two) or by other shortened rubric. When it was clear that
Access to the peroxo states through radiolytic reduction of oxygenated heme proteins
Even though it was appreciated in the mid-1970s that a peroxo state existed following a second electron transfer, it was not until the pioneering work of Davydov, Huttermann, Peterson, and others who had introduced pulse radiolysis and low-temperature radiolytic reduction of frozen heme proteins together with electron paramagnetic resonance characterization to define a doubly reduced dioxygen state [13], [14], [15], [16], [17], [18].
Radiolytic reduction at cryogenic temperatures is an approach
Spectroscopic characterization of a hydroperoxo state in P450 catalysis
Following the initial observation of an EPR signal consistent with a peroxo oxygen bound to the P450 heme via low-temperature radiolysis, there ensued a concerted effort to precisely define the electronic structure of the peroxo states by a variety of spectroscopic tools. Low-temperature stabilization of the ferrous–oxy complex, followed by introduction of the second electron via radiolysis, has allowed a detailed characterization of these intermediate states in P450cam CYP101. Although 60Co
EPR and ENDOR definition of P450 peroxo intermediates
Continued pioneering work by Davydov in collaboration with the EPR and ENDOR expertise of the Hoffman laboratory has provided an unprecedented look at the proton configuration associated with the heme-bound peroxide states in cytochrome P450 [21], [23], [30]. The application of 1H ENDOR to the cryogenically irradiated wild-type and mutant CYP101 oxy-complexes has enabled the visualization of proton(s) coordinated to ferric-peroxo species. This has allowed the delineation between a proton that
X-ray absorption spectroscopy to define P450 peroxo structures
The EPR and optical characterization of radiolytically prepared peroxo–ferriheme states have clearly provided an indispensable fingerprint of these intermediates in the P450 monooxygenases. However, despite an ever-growing database, detailed information about the peroxo geometry in heme enzymes has trailed behind that obtained for the non-heme iron inorganic counterparts [31]. Clearly required is a precise structural definition of the heme iron coordination environment and associated changes as
Raman spectroscopic characterization of heme–peroxo intermediates
While EPR/ENDOR can illuminate the existence of nearby protons potentially involved in oxygen activation and substrate functionalization, and XAS can precisely measure iron–nuclei distances, these techniques cannot define the electrostatics of the bound peroxo and separate the subtle differences between a short, strong hydrogen bond from a full covalent protonation event. Clearly needed are spectroscopic measurements of the relevant vibrational modes and energies of these intermediates. We have
Defining distal pocket hydrogen bonding and proton delivery
The unambiguous observation and spectroscopic characterization of the two-electron reduced peroxo–heme states in cytochrome P450 immediately raise the question as to the detailed three-dimensional structures of these states and an understanding of the distal pocket hydrogen bonding and proton-donating residues which contribute to the key steps in monooxygenase catalysis.
A major step forward in understanding P450 catalysis came through the use of site-directed mutagenesis of the bacterial P450
Conserved residues in the P450 active site
By the mid-1980s, there were several amino sequences of P450 cytochromes known, and the realization that this class of oxygenases was indeed only the tip of a very large superfamily. Currently, over 5000 nucleotide sequences have been postulated to code for cytochromes P450. Hence, for some time, it was natural to examine the alignment of predicted protein sequences to shed light on important catalytic residues. Earlier alignment of these sequences revealed two residues that were conserved
Structural characterization of distal pocket hydrogen bonding and catalytic processing
A major breakthrough in the understanding of P450 catalysis resulted from the X-ray structure determination of the ferrous-oxygenated state by Schlichting et al. [49]. Of critical importance were the structural changes that occurred upon oxygenation of the ferrous heme, Fig. 4. Immediately obvious was a “flip” in the backbone amide linkage between the pseudo-conserved acid (D251) and alcohol (T252) residues. This relocation of the carbonyl, now providing a hydrogen bond with N255, provides a
Understanding the uncoupling of electron flow in P450 oxygenases
The reaction cycle of Fig. 2 identifies several places where the channeling of reducing equivalents toward the cleavage of the heme-bound dioxygen can go astray. An obvious site for electron leakage to air is at the protein–protein interfaces between reductase, redoxin, and cytochrome P4450. Indeed, the autoxidation of flavin and iron–sulfur proteins has been well documented. Following transfer into the heme center, there remain three places where uncoupling can occur. The first is through
Summary
Thirty years of microbial cytochrome P450 work have dramatically increased our fundamental chemical and physical understanding of oxygenase function. Despite these advances, much still remains to be understood as to how Nature controls the formation and ultimate chemical reactivity of the intermediate states of metal, oxygen, and substrate to effect efficient catalytic processing. We look forward to summarizing additional advances on the celebration of the next decade of Professor Hayaishi’s
Acknowledgments
Our work over the past 30 years on the mechanistic enzymology and bioinorganic chemistry of P450 oxygenase catalysis has been generously supported by the National Institutes of Health. We are particularly thankful for a MERIT award (GM31756) that continues to provide the funds to define ever more precisely the functioning of P450 oxygenase systems. XAS measurements were made possible by the close collaboration of the Dupont-Northwestern-Dow CAT at the Advanced Photon Source and supported by the
References (51)
- et al.
A specific role of reduced adrenodoxin in adrenal mitochondrial steroid hydroxylases
Biochem. Biophys. Res. Commun.
(1971) - et al.
Pseudomonas putida cytochrome P-450. Characterization of an oxygenated form of the hemoprotein
Arch. Biochem. Biophys.
(1972) - et al.
P 450cam: oxygenated complexes stabilized at low temperature
Biochem. Biophys. Res. Commun.
(1977) - et al.
On the mechanism of compound I formation from peroxidases and catalases
J. Theor. Biol.
(1977) Intermediate spin-states in one-electron reduction of oxygen–hemoprotein complexes at low temperature
FEBS Lett.
(1979)- et al.
Electron spin and electron nuclear double resonance of the [FeO2]− [ferrite] center from irradiated oxyhemo- and oxymyoglobin
Biochim. Biophys. Acta
(1985) - et al.
Spin-density distribution in the [FeO2]-complex. Electron spin resonance of myoglobin single crystals
Biochim. Biophys. Acta
(1986) - et al.
EPR-spectroscopy of reduced oxyferrous-P450cam
FEBS Lett.
(1991) - et al.
Coming to grips with reactive intermediates
Adv. Inorg. Chem.
(1998) - et al.
Studies on the conformational changes of metalloproteins induced by electrons in water–ethylene glycol solutions at low temperatures. Haemoglobin
FEBS Lett.
(1974)
Cryotrapped reaction intermediates of cytochrome P450 studied by radiolytic reduction with phosphorus-32
J. Biol. Chem.
Cryoradiolysis for the study of P450 reaction intermediates
Meth. Enzymol.
Characterization of the oxygenated intermediate of the thermophilic cytochrome P450CYP119
J. Inorg. Biochem.
Cryogenic absorption spectra of hydroperoxo-ferric heme oxygenase, the active intermediate of enzymatic heme oxygenation
FEBS Lett.
Formation and decay of hydroperoxo–ferric heme complex in horseradish peroxidase studied by cryoradiolysis
J. Biol. Chem.
Electronic structures of active sites in electron transfer metalloproteins: contributions to reactivity
Coord. Chem. Rev.
Atomic resolution crystallography and XAFS
Coord. Chem. Rev.
Endogenous cysteine ligation in ferric and ferrous cytochrome P-450. Direct evidence from x-ray absorption spectroscopy
J. Biol. Chem.
P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli
Biochem. Biophys. Res. Commun.
Role of Thr-252 in cytochrome P450cam—a study with unnatural amino-acid mutagenesis
Biochem. Biophys. Res. Commun.
A role for Asp-251 in cytochrome P-450cam oxygen activation
J. Biol. Chem.
Superoxide anion production by the autoxidation of cytochrome P450cam
Biochem. Biophys. Res. Commun.
On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction
J. Biol. Chem.
Mechanism of the pyrocatechase reaction
J. Am. Chem. Soc.
Oxygen and life. Oxygenases, Oxidases and Lipid Mediators, International Congress Series
Cited by (76)
Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis
2023, Journal of Biological ChemistryAmbient O<inf>2</inf> is a switch between [1-electron/1-radical] vs. [2–electron] oxidative catalytic path in Fe-Phthalocyanines
2020, Chemical Physics LettersCitation Excerpt :These two reaction paths correspond to 2- or 1-redox equivalents above the FeIII state of FePcs [2]. Such a dual reaction path, two-electron or one-electron, has been also documented for cytochrome P-450 which can adopt either the FeIVO or [FeIV = Ο-Radical+] path, depending on the local environment of the Fe-heme [18,19]. However, so far, a convincing mechanistic study of the possibility to switch between the [1- or 2- redox equivalent] paths i.e. [FeIVO] or [FeIVΟ-Radical+], for the case of Fe-Pc is still lacking.
Spectroscopic studies of the cytochrome P450 reaction mechanisms
2018, Biochimica et Biophysica Acta - Proteins and ProteomicsCitation Excerpt :The electronic absorption spectrum of peroxo species has been obtained by cryoradiolytic reduction of oxyCYP101A1 D251N mutant that contains a perturbed proton delivery network in its active site, and exhibits the absorption maximum blue shifted by 2–3 nm with respect to the hydroperoxo intermediate (Fig. 9). The results of these and subsequent investigations have been summarized previously [38,39,350] and show the UV–Vis characterization of the peroxo and hydroperoxo states generated by low-temperature radiolysis of the ferrous-oxy complex in CYP101A1 [59,75,77], CYP119A1 [252], CYP3A4 [351] and chloroperoxidase [352] among other systems. The intermediates generated by one-electron reduction of diamagnetic oxy adducts of heme proteins are paramagnetic making electron paramagnetic resonance (EPR) spectroscopy especially valuable in characterizing the peroxo and hydroperoxo states of cytochromes P450 [15].
Mechanistic studies on versatile metal-assisted hydrogen peroxide activation processes for biomedical and environmental incentives
2016, Coordination Chemistry ReviewsRedox cycling in the activation of peroxides by iron porphyrin and manganese complexes. 'Catching' catalytic active intermediates
2016, Coordination Chemistry ReviewsCitation Excerpt :Studies on the intimate details of the catalytic cycle of these enzymes have attracted significant attention from bioinorganic scientist, not only due to their biological relevance, but also due to the desire to design powerful catalysts with a high reactivity potential. Special efforts have been put into structural and electronic characterization of the iron(IV) oxo porphyrin π-cation radical, termed Compound I, that is perceived to be responsible for the demanding activation of CH bonds [1–9]. The highly reactive Compound I, well characterized for peroxidases [10–14], was postulated to also be a crucial intermediate in the cytochrome P450 catalytic cycle.