Chemical models of enzymes' active sites aid our understanding of biological reactions. Such a model of a reaction intermediate promises to advance our knowledge of the biochemistry of iron-containing haem enzymes.
Reporting in Angewandte Chemie, Liu et al.1 describe a tractable and functional chemical model for a reaction intermediate that is central to a variety of biologically important processes. Their model, known as a ferric hydroperoxo–haem intermediate, is chemically analogous to intermediates that have been observed or proposed for several classes of enzymes that oxidize substrates using molecular oxygen. The authors' discovery represents a considerable advance in biomimetic chemistry, and should help to resolve vital mechanistic questions about the biochemistry of these indispensable enzymes2,3.
Haems are chemical groups that consist of an organic, ring-shaped compound (known as porphyrin) with an iron atom bound at its centre (Fig. 1a). They are found in the active sites of many proteins that fulfil a diverse range of biological functions, including metabolic oxidation reactions and the transportation of diatomic gases, such as oxygen. The iron atom acts as a source or sink of electrons for redox reactions, and is the anchor to which diatomic molecules bind. In some enzymes, the porphyrin ring also acts as an electron source.
The most common haem in nature, protoporphyrin IX (Fig. 1a), is found in the oxygen-transport and storage proteins haemoglobin and myoglobin. In these proteins, the haem iron atom binds to one of the atoms in oxygen to form an oxy compound (Fig. 1b). Oxygen-activating enzymes take things further: once an oxygen molecule is bound, the resulting oxy complex is reduced — a single electron is transferred from the iron atom to the bound oxygen molecule — to produce a ferric peroxo species and/or a ferric hydroperoxo complex (Fig. 1b). These complexes are thus central to our understanding of haemoprotein reaction mechanisms, so it is highly desirable to generate models of the complexes, characterize them and study their reactivity.
The generation and characterization of enzymatic hydroperoxo intermediates has previously been achieved by reducing oxy precursors at low temperatures (typically below 77 K)4,5. Liu and colleagues1 now report the preparation of a hydroperoxo complex (Fig. 1c) either from a synthetic analogue of oxy or from a related ferric peroxo species. The authors thoroughly characterized their complex using a panel of spectroscopic techniques, and showed conclusively that it was the desired ferric compound — an accurate biomimic that now awaits further study. This represents a crucial advance over earlier synthetic models of haemoprotein reaction intermediates.
To which proteins and protein functions might Liu and colleagues' biomimetic compound be relevant? Ferric hydroperoxo species are formed in the peroxidase and catalase classes of enzyme6, where they are often referred to as compound 0 (cpd 0, Fig. 1b). The hydroperoxo intermediates are precursors to more highly oxidized iron–oxygen intermediates, known as cpd I and cpd II, which are the active intermediates in many haem-containing enzymes. Peroxidases and catalases cleave the oxygen–oxygen (O–O) bond in hydrogen peroxide (H2O2). Both types of enzyme remove H2O2, which is a potentially harmful by-product of many metabolic processes. Catalases detoxify H2O2 by converting it into oxygen and water; peroxidases use it to oxidize other substrates, leaving only water as a by-product.
An O–O cleavage process starting from a cpd 0 entity is also thought to be key to the cytochrome P450 monooxygenase superfamily of enzymes7,8,9. These enzymes effect the addition of one oxygen atom — derived from the oxygen atom in cpd II — to a biological substrate. Cytochrome P450 enzymes have various metabolic functions, such as the biosynthesis of steroid hormones, vitamins and antibiotics, and are crucial for initiating the removal of potentially toxic xenobiotics ('foreign' compounds in organisms). One subgroup of the cytochrome P450s is the aromatases, which convert the steroid androstenedione to the hormone oestrone. This involves three successive oxidations, each requiring oxygen and NADPH (a naturally occurring reducing agent). The first two oxidations are thought to be mediated by a cpd I entity, whereas the third involves a ferric peroxo compound10,11. Because aromatase activity and oestrone levels are elevated in most breast cancers, the enzymes are potential drug targets.
Nitric oxide (NO) synthases2,12,13 provide interesting examples of how nature can adapt haem-containing active sites to very different roles. As in cytochrome P450s, the active sites of NO synthases contain a haem bound to a cysteine amino acid (the base, B, in Fig. 1b). But their activity is confined to the amino acid L-arginine, which it converts to NO — a signalling molecule vital to the nervous, immune and cardiovascular systems. The chemistry involves two sequential oxidations, each requiring oxygen, protons and NADPH. Each step proceeds via oxy, and follows on to either peroxo2, hydroperoxo or cpd I intermediates.
In some enzymes, such as haem oxygenases (HOs), ferric hydroperoxide is the oxidizing species14, and the substrate is the haem itself. HOs are found in many organisms, and in mammals the oxidation products are biologically vital: biliverdin, which acts as an antioxidant; liberated iron(II) ions, which are recycled for use elsewhere (primarily in haems); and carbon monoxide, which is used as a neurotransmitter. Reactions mediated by the enzyme cytochrome c oxidase, a member of the haem–copper oxidase (HCO) superfamily, probably also proceed through a ferric hydroperoxo complex, which then undergoes O–O cleavage and formation of cpd II (ref. 15). HCOs facilitate proton pumping across mitochondrial membranes, which generates a proton gradient that is used in the formation of ATP, the cell's energy carrier.
The enzymes discussed above exemplify the extensive involvement in biology of the reaction pathway proceeding from oxy to cpd I and/or cpd II species, through ferric hydroperoxo complexes. The occurrence and precise control of the active-site chemistry in this pathway are essential for proper biochemical functioning. For example, considering the role of ferric hydroperoxo complexes in HOs, improper processing of the reactions in the pathways of other enzymes could lead to unwanted haem degradation. It is therefore crucial to understand fully the manner in which biological hydroperoxo species form, react with different substrates, become protonated and undergo O–O cleavage2,7,8,9, and to learn the order of these events2.
The generation of well-described model compounds such as that reported by Liu et al.1 will enable fundamental insights into haem-enzyme reactions, intermediates and mechanisms to be obtained from chemical investigations9. Such biomimetics may also shed light on a vast range of other enzymes that use oxygen, whose reactions are closely related to those of their haem-containing siblings3.
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