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Sawyer^

Introduction to Oxygen

Activation

The cytochromes P450 have occupied a central

focal point in biochemistry, pharmacology, toxicol-

ogy, and biophysics for more than three decades.

This rich history is faithfully conveyed in accom-

panying articles appearing in this volume as well as

those of the first and second editions of

Cytochrome

P450 edited by Ortiz de Montellano^'

In addition,

there have been innumerable review articles and

book chapters devoted to the subject^'

Interest in

the cytochromes P450 perhaps began due to the

importance of these catalysts in human physiology

and pharmacology. The interest of chemists and

biophysicists peaked due to the many unique spec-

tral features of these heme enzymes. One of the

most intriguing aspects of P450 systems is the

range of difficult chemistry efficiently catalyzed.

the importance of oxygen as a cosubstrate, and the

thoughts of overall common mechanisms with the

heme monooxygenases and other systems that

effect reductive metabolism of atmospheric dioxy-

gen^'

Thus, from the very beginning, the phrase

"active oxygen" was used to label a holy grail of

cytochrome P450 research: the documentation

of the intermediate states of oxygen and heme

iron that give rise to the diversity of metabolic

chemistries observed.

The chemistry of "oxygen activation" cat-

alyzed by a variety of metal centers has a long and

rich history, and as many as eight variations of

structurally different metal-oxygen complexes

were discussed as early as

1965^.

This publication

of Cytochrome P450, Third Edition is particularly

timely. During the past 3-5 years, a great deal of

progress has been realized in the direct observa-

tion of

P450

intermediate states and the evolution

of a detailed understanding of the structural

Thomas M. Makris • Center for Biophysics, University of

Illinois,

Urbana, IL.

Ilia Denisov • Department of

Biochemistry,

University of

Illinois,

Urbana, IL.

lime Schlichting • Max Planck Institute for Medical Research, Department of Molecular

Mechanisms,

Germany.

Stephen G. Sligar • Departments of Biochemistry, Chemistry, the School of Medicine, and the Center for

Biophysics, University of

Illinois,

Urbana, IL.

Cytochrome

P450:

Structure,

Mechanism,

and

Biochemistry,

3e, edited by Paul R. Ortiz de Montellano

Kluwer Academic / Plenum Publishers,

New

York,

2005.

149

150

Thomas M. Makris ef al.

features of the protein, which control the popula-

tion and stepwise movement through the reduction

and protonation states of heme and oxygen. The

veil surrounding "oxygen activation" has been

lifted through the systematic application of

vari-

ety of biochemical and biophysical tools, such as

mutagenesis, high-resolution cryocrystallography,

multiparameter spectroscopic studies of interme-

diate states isolated using cryogenic or fast kinetic

techniques, and many rigorous quantum chemical

and molecular dynamics computational studies.

Equally important has been the explosion of

genomic and proteomic breakthroughs. No longer

are we dealing with a single microbial P450 and a

few membrane bound mammalian counterparts.

Rather, with over 3,000 P450 genes known, we

can find examples from unique environmental

niches where the enzymatic reaction cycle yields

newly observable features. Thus today, the current

view of oxygen activation mechanisms catalyzed

by metal centers in heme enzymes ensures a much

better opportunity to develop recurring catalytic

paradigms than was possible at the time of the

Cytochrome P450, Second Edition.

A broader view of heme-oxygen catalysis with

the understanding of how proximal ligand, protein

structure, and subtle control of electron/proton

stoichiometry contribute to very different reactiv-

ities is now within reach^. Common mechanistic

hypotheses for oxygen activation in metalloen-

zymes are currently emerging. Recent progress in

mechanistic studies on other heme enzymes,

which use different forms of so-called "active

oxygen intermediates," such as peroxidases'^' •',

heme oxygenases (HOs)'^' '^, catalases'"^, nitric

oxide synthases (NOS)'^, and peroxygenases'^' '^,

have provided an impressive degree of unifica-

tion. A fundamental question that has always

plagued the interpretation of experimental data

has been the functional differentiation of these

enzymes, which efficiently catalyze a range of

diverse chemistries yet utilize similar highly reac-

tive heme-oxygen complexes. The comparison of

similar reactive intermediates in different enzymes

has helped to distinguish between the essential fea-

tures of each of the enzymes. This in turn has pro-

vided important clues into the role of protein

structure in the heme-oxygen catalytic events. The

recent progress in actually observing, through

kinetic isolation and cryogenic techniques, these

fundamental intermediate states in the cytochrome

P450s brings us to a much deeper understanding of

P450 monooxygenase catalysis that was available

at the time of the previous edition of this volume.

In discussing the reaction cycle of the

cytochrome P450s, it is useful to incorporate

similar states which appear to be more or less

stable in other heme-oxygen systems. The sequen-

tial steps of "oxygen activation" are those that

follow the formation of the ferrous reduced iron-

porphyrin state that is generated by electron

transfer from the physiological redox donor. These

steps are formally:

Dioxygen binding to the ferrous heme

center to form an oxygenated complex which can

be written as either Fe^^-OO or Fe^^-00~.

One-electron reduction of this complex

to form the Fe^^-00^~, ferric-peroxo complex.

Protonation of the distal oxygen of

this peroxo anion to generate the Fe^^-OOH~,

ferric-hydroperoxo complex.

A second protonation of the Fe^^-OOH~

complex at the distal oxygen atom to form a

transient oxo-water adduct which immediately

undergoes heterolytic scission of the 0-0 bond.

This process generates a single oxygen atom con-

taining iron-porphyrin species, which is formally

two redox equivalents above the ferric heme start-

ing state. This intermediate has been variously

termed the "iron-oxo," "ferryl-oxo porphyrin

TT-cation radical," or "Compound I," the latter after

the pioneering discoveries made by Theorell,

Keilin, George, and Chance while studying the

peroxidases and catalases'^.

The intermediate states so defined have coun-

terpoints in nonheme oxygen activation and have

been also studied and reviewed in numerous arti-

cles over past decades. We note that a search of the

term "oxygen activation" in SciFinder Chemical

Abstracts Database returns almost 29,000 refer-

ences published after 1995! Thus, we will not

attempt to link nonheme iron chemistry, nor

oxygen activation in multicenter metalloenzymes

which was recently reviewed by Siegbahn'^, with

that of the P450 systems in this chapter. Rather,

we focus on those features which directly address

the important aspects of the oxygen activation

cascade in P450. The structure and properties of

relevant porphyrin complexes, including models

of cytochrome P450 and other oxygen-activation

enzymes, are extensively reviewed in the recent

edition of

the

Porphyrin Handbook^^'^^.

Activation of IVIolecular Oxygen by Cytochrome P450

151

General Features of Dioxygen

Activation in Heme Enzymes

As discussed in the first section of this chapter,

the P450 cytochromes, NOS, HOs, and cyto-

chrome oxidases are all heme enzymes which are

able to "activate" molecular oxygen. Related

heme enzymes, such as the peroxidases and cata-

lases,

work with partially reduced dioxygen

species. It has always been intriguing that each

of these enzymes uses the same cofactor, heme

or Fe-protoporphyrin IX, but performs a distinct

physiological function.

In heme enzymes that are able to activate

molecular dioxygen, the porphyrin and proximal

ligand provide the heme iron with five coor-

dination sites. Thus, the side-on coordination of

peroxide, which is common for nonheme metal-

loenzymes and oxygen activation mechanisms, is

not usually observed in heme enzymes and is also

rare in model metalloporphyrins^^'

^^~^^.

A common role of the porphyrin ring in oxygen

activation is the donation of an electron during the

overall process of O-O bond cleavage^^. The rela-

tively easy oxidation of Fe-protoporphyrin IX,

which serves as an additional buffer supply of

electrons to the dioxygen ligand, is a common

feature in the mechanisms of related heme enzyme

families, such as peroxidases, heme catalases,

cytochromes P450, NOS, and peroxygenases,

which catalyze 0-0 bond cleavage of a peroxide

ligated to the heme iron^^'

Donation of one elec-

tron from the porphyrin to the peroxide ligand ini-

tiates the effective heterol3^ic scission of the O-O

bond and results in formation of a cation radical

on the porphyrin ring, a definitive feature of a

"Compound I" state^^

Oxygen activation in all of these systems

begins with the binding of dioxygen as an axial

ligand to the Fe^+ heme iron, or with binding

H2O2

to the Fe^^ heme. Thereby hydroperoxo-

ferric heme complex is formed through different

pathways as a common intermediate state, shown

in Figure 5.1. Thus, a current focus of research

is this common "peroxo," both in the pathways

for its formation as well as in the control of

Oxidase ©

Heme Oxygenase ®-

Peroxygenase ©

-MD

Compound

ferrous dioxygen

02^J

Nucleophiiic

Oxidations

peroxo-mmon

N N

/ Fei"/L.

Etectrophiiic

Nucleophlfic

Oxidations

/ Fe^v/

N f-~n

N-AH2

ferric hydroperoxo Compound i

N f-N

^' »X

Fell'/—^

H9O,

Fe^

N f-N

N4J

h-N

H,0 tl

2e .

2HU

rvH:>o

Radical

Oxidations

Figure 5.1. Common reactive intermediates in bioinorganic catalysis. The catalytic pathways of various heme

enzymes are shown in the inset.

152

Thomas M. Makris et al.

proton delivery events, which dictate the inherent

reactivity of the resulting "active oxygen" inter-

mediates. In the classic P450 reactivities of a

"Compound I like" intermediate, it is also this

critical acid-base chemistry which initiates dioxy-

gen bond scission to form the higher valent

iron-oxo state.

In addition, there are other redox active

pathways operant in the cytochromes P450

(Figure 5.1). First is an "oxidase" pathway using

atmospheric dioxygen and two additional redox

equivalents in addition to the two required for

heterolytic 0-0 bond cleavage. Additionally there

is a "peroxidase/peroxygenase" pathway wherein

hydrogen peroxide serves as an oxygen donor.

A detailed analysis of these pathways with respect

to oxygen activation in the cytochromes P450

was provided in earlier editions of

this

volume^' ^.

Obviously, these two mechanisms differ by the

overall redox state of oxygen vs peroxide and the

need in two additional reduction steps (one-

electron reductions by the physiological redox

partner) in the oxidase pathway. Perhaps most

important, however, is the difference in the proto-

nation states that necessitate specific delivery of

two protons to a bound peroxo intermediate when

beginning with dioxygen as opposed to a 1-2

proton shift to form the transient Fe-0-0H2 pre-

cursor when starting with hydrogen peroxide and

ferric heme.

2.1.

The Oxidase/Oxygenase

Pathway in Cytochrome

P450

The pivotal role of different protonation path-

ways to bound oxygen derivatives in bioinorganic

chemistry has been shown in various mechanistic

studies ^^' ^^' ^^. The same concept has also been

considered for the case of catalysis involving

H2O2,

in which the correlation between the ability

to efficiently deliver the proton to the metal-

bound hydroperoxide anion, and the efficiency of

catalytic decomposition of hydrogen peroxide has

been noted^'*. In heme-enzyme catalysis, however,

the ability to supply the necessary one or two

protons for the generation of the proper active

intermediate from the iron-bound peroxide/

hydroperoxide anion state is an essential and

crucial aspect of the detailed structural "fine-

tuning" provided by the enzyme active center.

Thus,

the relative spatial orientation of catalytic

residues and active-site water molecules may

facilitate or suppress various branches of the

microscopic pathways that contribute to the possi-

ble chemical reactions involving these activated

oxygen compounds. An encompassing scheme

that graphically illustrates the commonality of

these various processes is depicted in Figure 5.1.

Many laboratories have focused on the role of

protein structure in defining the pathways and

control points outlined in Figure 5.1. Recent

results, which are discussed in this chapter, have

greatly improved the understanding of these

events. For example, a hydrogen-bond network

has been implicated as the critical component of

the enzymatic mechanism which defines both cat-

alytic activity and the effective branching points

between productive and nonproductive pathways

for the camphor hydroxylation catalyzed by

cytochrome P450 CYPIOP^-^^ Modification of

this network, either through mutagenesis of the

CYPlOl enzyme, or by the use of substrate

analogs, points to the subtle active-site features of

the protein, which can in some cases have dire

consequences on the productive monooxygenase

pathway with resultant abortive processes that

short circuit the classic P450 mechanistic "wheel"

(Figure 5.2). These additional pathways, since

they bleed redox equivalents and dioxygen from

the pure monooxygenase stoichiometry, have been

termed "uncoupling" events. While the mecha-

nisms of uncoupling may have either a thermody-

namic and/or kinetic origin, it is most certainly

governed by the role of a protein-provided "proton

relay" system. These pathways of proton delivery

can involve not only backbone and side-chain

moieties from the protein structure, but also local-

ized water molecules, which are appropriately

stabilized in the active center of the enzyme.

The realization of how Nature uses specialized

water alludes to analogous discussion of proton

delivery in diverse enzymes such as cytochrome

oxidase"^^' ^^, and introduces these concepts into

the vernacular of P450 specialists. Again, geno-

mics and proteomics have helped identify critical

structural features, for instance the famous "acid-

alcohol pair," Asp251-Thr252 in the cytochrome

P450 CYPIOI. Only most recently, however, has

structural information shed critical light on how

these residues work in concert to deliver protons

at the right place and time.

Activation of Molecular Oxygen by Cytochrome P450

153

Figure 5.2. The P450 catalytic cycle.

An oxidase mechanism of oxygen activation

supported by NADH has also been suggested as

an alternative activity in the peroxidases, but was

later disproved"^^. Both the cytochromes P450 and

peroxidases are thought to share a high-valent

"ferryl-oxo" porphyrin cation radical intermedi-

ate^^'

^^.

In P450 catalysis, atmospheric dioxygen

binds to the reduced heme iron of the enzyme,

and this ferrous-oxygen, or ferric-superoxide

complex accepts one more electron from its

protein redox partner to form the peroxo-ferric

intermediate. This in turn is quickly protonated to

generate the hydroperoxo-ferric intermediate"*^'

^^.

The formation of this intermediate from the ferrous-

oxy complex, and its subsequent transformations

were recently investigated in detail using the

method of cryogenic radiol3^ic reduction"*^'

^^'

^^.

The same iron-hydroperoxo intermediate is

assumed to be a common state in the formation of

"Compound F' in peroxidases and a variety of

other systems such as the HOs^^'

^^' ^^~^^.

Contrary

to P450 and HO, in peroxidases the natural for-

mation of this active intermediate involves the

hydrogen peroxide starting material. In contrast to