Substrate Oxidation by Cytochrome P450 Enzymes

6 Substrate Oxidation by Cytochrome P450 Enzymes Paul R. Ortiz de Montellano and James J. De Voss 1. Introduction P450^^^ (CYPlOl)io, P450B^.3 (CYP102)11' '\ P450^^^p (CYP108)i3, P450^^yp (CYP107Al)l^ The cytochromes P450 are catalytic hemo- P450^^^ (CYP55Al)i^ Sulfolobus solfataricus proteins in which the heme iron atom is coordi CYPl 19^^ Streptomyces coelicolor CYP154Cli\ nated to a proximal cysteine thiolate. This thiolate Mycobacterium tuberculosis CYP52^^, Sorangium ligand is responsible for the characteristic Soret cellulosum P450epoK^^, and the mammalian absorption maximum of the Fe"-CO complex at CYP2C5 (see Chapter 3>f^. Although the thiolate —450 nm and is critical for P450 catalysis^. Early ligand is always present, the distal water ligand site-specific mutagenesis studies with CYP1A2 appears to be absent in some mammalian and P450^^j^ suggested that replacement of the enzymes, either because the water does not bind in cysteine thiolate by a histidine ligand gave inac those structures or because it is displaced by an tive protein^' ^. Detailed studies of the P450^^^ endogenous ligand^ ^ Cys357His mutant have recently confirmed that The c)^ochrome P450 catalytic cycle is initiated this mutant enzyme has an almost undetectable by the binding of a substrate, usually with con catalytic activity^' ^. The low camphor-oxidizing comitant displacement of the distal water ligand. activity is paralleled by a low rate of reduction of The ferric heme is then reduced to the ferrous state the iron, an elevated autooxidation rate, and an using electrons provided by suitable electron donor observable peroxidase activity"*. The thiolate lig proteins (see Chapter 4). In cytochrome P450^^ and is thus clearly critical for P450^^j^ function, and many other P450 enzymes, substrate binding although the relative contributions of the elec is widely believed to be a prerequisite for the trans tronic vs structural perturbations of the mutation fer of the first electron to the iron, but in some to the low catal)^ic activity remain unclear. These enzymes electron transfer can occur without the results agree with the results of experiments with prior binding of a substrate^ ^. Reduction of the iron thiolate ligated metalloporphyrin model systems^' ^ is followed by binding of oxygen to give the fer and of computational analyses of the role of the rous dioxy complex. Transfer of a second electron thiolate (see Chapter 2)^' ^. to this complex produces the ferric peroxy anion The heme iron ligand on the distal side is a (PorFe"^-00~, where Por = porphyrin) or, after water molecule in all the available crystal struc protonation, the ferric hydroperoxo complex tures of substrate-free P450 enzymes, including (Por"^-OOH) (Figure 6.1). Heterolytic cleavage of Paul R. Ortiz de Montellano • Department of Pharmaceutical Chemistry, University of California, San Francisco, CA. James J. De Voss • Department of Chemistry, University of Queensland, Brisbane, QLD Australia. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3e, edited by Paul R. Ortiz de Montellano Kluwer Academic / Plenum Publishers, New York, 2005. 183 184 Paul R. Ortiz de Montellano and James J. De Voss |Fe"-02|[sH]--^02 yj+ f 1 H ' |Fe=o]|sH]-^--— |Fe"i-02H||sH]-*— [Fe"'-02-l[sH] —rl20 • 2e- t t H2O H2O2 Figure 6.1. The general catalytic cycle of cytochrome P450 enzymes. The [Fe"^] stands for the resting ferric state of P450, and SH for a substrate molecule. The shunt pathway utilizing H2O2 is shown as are three sites for the uncoupling of the enzyme to give, respectively, O^, H2O2, or H2O. the dioxygen bond in this peroxo intermediate (b) a molecule of H2O2 dissociates from the ferric extrudes a molecule of water and forms the hydroperoxide complex, or (c) two electrons are putative ferryl oxidizing species (Figure 6.1). used to reduce the ferryl species to a molecule of Hydrogen bonding of the distal ferric hydroperoxo water before it can be used in a reaction with the oxygen, directly or via a water molecule, to a substrate (Figure 6.1). The parameters that govern highly conserved threonine facilitates this het- uncoupling at each of the three stages are unclear, erolytic cleavage (see Chapter 5)^^~^^. The ferryl but factors that contribute to uncoupling appear to species is thought to be responsible for most P450- be the degree of uncontrolled water access to the catalyzed oxidations, although the ferric peroxo active site, the extent to which the substrate can anion and the ferric hydroperoxo complex have reside at unproductive distances from the ferryl been invoked as oxidizing species (see below). It is species, and the presence or absence of sufficiently usually, but not always, possible to circumvent the reactive sites on the substrate molecule^^' ^'^-'^^, The requirement for activation of molecular oxygen in catalytic efficiency of a P450 enzyme can be seri a so-called "shunt" pathway by employing H2O2 or ously impaired by uncoupling, as evidenced by the some other peroxide as a co-substrate (Figure 6.1). contrast between nearly quantitative coupling in the However, the oxidizing species thus obtained is oxidation of camphor by P450^^j^ and a process that apparently not identical to that obtained by normal is more than 95% uncoupled when the same oxygen activation. Thus, peroxides cannot replace enzyme oxidizes styrene^^. A higher degree of molecular oxygen activation in some reactions, intrinsic uncoupling is often observed in mam they often give product distributions that differ sig malian P450 enzymes, some of which can be sup nificantly from those obtained by molecular oxy pressed by interaction of the P450 enzyme with gen activation^^"^^, and they cause a more rapid reduced cytochrome b^^^' ^^. degradation of the prosthetic heme group^'. The P450 oxidation stoichiometry requires one molecule of oxygen and two electrons from NAD(P)H to add one oxygen atom to a substrate. If 2. Activation of the ratio of reduced pyridine nucleotide (or oxygen) iVIolecufar Oxygen consumed to product formed is greater than one, the enzyme is said to be uncoupled. Uncoupling Cryogenic X-ray crystallographic, EPR, occurs when (a) the ferrous dioxy complex reverts ENDOR, and spectroscopic studies have convinc to the ferric state by dissociation of superoxide. ingly identified several intermediates in the P450 Substrate Oxidation by Cytochrome P450 Enzymes 185 catalytic cycle (see Chapter S)^^"^^. These include three substrates plus styrene"*^. To rationalize this the ferric, ferrous, ferrous dioxo, and ferric observation, the authors argued that hydroxylation is hydroperoxo complexes of P450^^j^. Crystallo- mediated exclusively by the ferryl whereas epoxida graphic evidence has also been reported for the tion can be mediated by both the ferryl and ferric ferry 1 species^^, but this intermediate has not been hydroperoxide intermediates. Thus, impairing for detected by other sensitive cryogenic approaches mation of the ferryl species by removing the cat and its attribution to the ferryl species remains alytic threonine would decrease hydroxylation but open to question. In low-temperature EPR, have little effect upon epoxidation. However, in con ENDOR, and spectroscopic studies, the ferric trast to the results with the CYP2E1 T303A mutant, hydroperoxide intermediate disappears as the the corresponding T302A mutant of CYP2B4 hydroxylated camphor product appears without exhibited both decreased hydroxylation and epoxi the observation of any intermediate species^^' ^^. dation rates. This discrepancy does not necessarily All the intermediates in oxygen activation by contradict the hypothesis, as it could reflect differ P450 have thus been observed except for the crit ential changes in the active sites of the two proteins ical ferryl species, which remains elusive and in addition to elimination of the hydrogen bond that undefined. facilitates ferryl formation, hi a more recent study in As already mentioned, the activation of molec which Thr252, the catalytic threonine of P450^^^, ular oxygen can often be circumvented if perox was mutated to an alanine, it was found that cam ides are used as activated oxygen donors. Efforts phor hydroxylation was suppressed, but the epoxi to identify the reactive oxygen species in these dation of an olefinic camphor analogue could still peroxide-supported reactions have been pursued be observed'*^. However, the epoxidation reaction for many years'^ ^^^. The species that has been spec- occurred at a much slower rate (<20%) despite the troscopically detected in these reactions has the expectation that the steady-state level of the ferric spectroscopic signature of a ferryl intermediate^^, hydroperoxide should be elevated. This finding is but evidence is lacking that this intermediate is the consistent with the prediction by computational same as that produced by the activation of molecu studies that the ferric hydroperoxo complex should lar oxygen. To the contrary, the reactions with per be a very poor olefin-oxidizing agent^^. These oxides have been shown to produce EPR signals results argue that in the wild-type proteins, the fer tentatively attributed to tyrosine radicals^ ^' '^^' ^^, but ric hydroperoxide makes no more than a small con no such radicals have been observed under normal tribution to epoxidation, and none to hydroxylation. turnover conditions. Furthermore, as noted earlier, In a second study, the iV-oxidation of amines by the peroxide-mediated reactions do not always CYP2B4 and its T302A mutant supported by either faithfully

Load more