Review
Thirty years of microbial P450 monooxygenase research: Peroxo-heme intermediates—The central bus station in heme oxygenase catalysis

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Abstract

Oxygen has always been recognized as an essential element of many life forms, initially through its role as a terminal electron acceptor for the energy-generating pathways of oxidative phosphorylation. In 1955, Hayaishi et al. [Mechanism of the pyrocatechase reaction, J. Am. Chem. Soc. 77 (1955) 5450–5451] presented the most important discovery that changed this simplistic view of how Nature uses atmospheric dioxygen. His discovery, the naming and mechanistic understanding of the first “oxygenase” enzyme, has provided a wonderful opportunity and scientific impetus for four decades of researchers. This volume provides an opportunity to recognize the breakthroughs of the “Hayaishi School.” Notable have been the prolific contributions of Professor Ishimura et al. [Oxygen and life. Oxygenases, Oxidases and Lipid Mediators, International Congress Series, Elsevier, Amsterdam, 2002], a first-generation Hayaishi product, to characterization of the cytochrome P450 monooxygenases.

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

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