(1) A large number of biological oxidation reactions use dioxygen, O2, and proceed through a step-wise cleavage of the two bonds of O2. The reversible reaction of O2 and haemoglobin is prototypical of a widespread phenomenon in which the forward reaction depends on the reductive cleavage of one of the O2 bonds to give a superoxide anion that then forms a hexacoordinated iron complex (see below). It is argued that this feature of haemoglobin represents a fundamental reaction which operates in all iron-containing oxidases; irrespective of whether they contain haem-iron as in P-450 enzymes or a non-haem iron as is present in prolyl and lysyl hydroxylases. Haemoglobin + O = O reversible Oxyhaemoglobin [GRAPHICS] (2) In P-450 dependent hydroxylases the initially formed Fe(III)-O-O. species is converted into Fe(III)-O-OH and the heterolysis of the second oxygen-oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe(V) = O <--> (+.) Fe(IV = O <--> Fe(IV)-O.. One of these, Fe(IV)-O. behaves like an alkoxy radical and participates in hydrogen abstraction from a C - H bond to produce Fe(IV)-OH and a carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe(IV)-OH. The latter species may thus be regarded as a carrier of hydroxyl radical. (3) Some P-450 systems, notably aromatase and 14-alpha-demethylase, catalyse not only the hydroxylation reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C-C bond cleavage process. All these reactions occur at the same active site. The conversion -CH2OH --> -CHO has been explained using the oxo-derivative, Fe(IV)-O.. (4) The C-C bond cleavage reaction conforms to the general equation shown below and may be rationalized by two alternative mechanisms one of which uses the oxo-derivative, Fe(IV)-O. and the other Fe(III)-OOH. The merits as well as the drawbacks of the two mechanisms are considered and our own bias for a mechanism involving Fe(III)OOH has been emphasized. [GRAPHICS] (5) The oxo-derivative is also involved in reactions with olefinic and aromatic double bonds and in our view these reactions are best rationalized by invoking that in these cases the electronic structure of the substrate promotes the oxo-derivative to reveal its electrophilic character [(+.) Fe(IV) = O <--> Fe(III)-O+]. (6) Catalase and peroxidases are also haem containing enzymes and these use a variety of peroxides as oxidants. Since in the peroxides one of the double bonds of the putative precursor O2 is already reduced, these react with the Fe(III) form of enzyme directly to produce the Fe(V)-OOH species that is converted to the oxo-derivative (Fe(V) = O <-> Fe(IV)-O.). The latter species then participates in the decomposition of H2O2, catalysed by catalase, or the dehydrogenation of AH2 catalysed by various peroxidases via a radical mechanism involving an initial hydrogen abstraction followed by disproportionation. (7) Cytochrome c oxidase is present in all aerobic organisms and catalyses the terminal 4 electron reduction of dioxygen into H2O. This reaction may be considered as a step-wise process in which 2 electrons are first used to convert O2 into the oxoderivative via the usual hydroperoxide species. The remaining 2 electrons are then used for the reductive cleavage of the oxoderivative into water. (8) Hydroxylation reactions normally associated with P-450 types of cytochrome are also catalysed by enzymes which contain non-haem iron, for example proline and lysine hydroxylases. In the resting states these enzymes contain Fe(II) and use alpha-oxoglutarate as the reductant. The mechanism of the hydroxylation reaction catalysed by these enzymes is similar to that of P-450 enzymes. Except that the stoichiometry of electrons involved dictates that compared to the haem containing P-450 enzymes the oxo-derivative in the non-haem enzymes may be at a higher reduction level, i.e. Fe(III)-O. instead of Fe(IV)-O.. (9) Another group of enzymes which have not yet been studied in detail are related to P-450 as well as to non-haem oxygenases. These like the former use NAD(P)H but like the latter contain non-haem iron. An oxygenase of this type could be involved in the oxidation of the 4-alpha-methyl group of sterols to the corresponding carboxylic acid. It is hypothesized that the enzymes of this class may also catalyse the hydroxylation reaction through the participation of Fe(IV)-O. as described in (2), except that in this case all the five ligands for the coordination of iron are provided by amino acid side chains. (10) The desaturation process, -CH2-CH2- --> -CH = CH-, CH-, requires NAD(P)H and 02 and is also catalysed by enzymes containing non-haem Fe(III) as in (9). This transformation may be envisaged to represent an alternative reaction course in which the neutralization of the two radical species (Fe(IV)-OH and the carbon radical) is achieved, not by an associate reaction, as in hydroxylation, but through disproportionation. (11) It is argued that the stereochemical control exercised by P-450 and related enzymes, in which the retention of stereochemistry is generally observed in hydroxylations and the removal of cis-oriented hydrogen atoms in desaturations, are not the consequences of the reaction mechanism itself. These are, however, due to the constraints on the mobility of the substrate within the Michaelis complex. It makes sense to assume that enzyme systems operating through the intermediacy of particularly reactive species such as free radicals have evolved to allow a minimum motion of bonds during catalysis, in order to ensure that the species do not participate in random reactions and damage the decor of the active site.
