Other Routes to Driving P450 Catalytic Function

The fact that most P450 enzymes require at least one redox partner protein is often seen as a complicating factor in their exploitation for biotechnological applications. However, there are means to drive P450 catalysis that do not rely on exogenous protein partners. The best known of these is the peroxide shunt mechanism (as illustrated in Fig. 5.2) whereby the resting state of the P450 can be converted to the ferric hydroperoxy form by reaction with hydrogen peroxide (or organic peroxides). This is the reversal of the uncoupling reaction that leads to collapse of the ferric hydroperoxy species. Protonation of this species creates the reactive compound I, although the ferric hydroperoxy state itself may also participate in certain P450-dependent reactions [18, 120]. In theory, the peroxide shunt represents a cheap alternative to use of expensive nicotinamide coenzymes and multiple protein partners. However, the reality is that peroxides oxidatively degrade the heme macrocycle and attack amino acid side-chains. Thus, P450 inactivation appears an inevitable consequence of exploitation of the peroxide shunt method, and this has prevented its widespread application for chemical transformations.

Recent mutagenesis studies have aimed to address the peroxide instability issue, and have generated P450BM-3 mutants with moderately increased activity (but not stability) with peroxide [121]. Perhaps inevitably, nature has found more appropriate structural solutions to the problem, and B. subtilis P450BSp (CYP 152A1) is a peroxygenase P450 that has evolved to use hydrogen peroxide directly to facilitate hydroxylation of fatty acids [122]. Homologs of this P450 have been recognized in other organisms (e.g. Sphingomonas paucimobilis, CYP152B1), and analysis of its atomic structure will clearly provide important clues as to adaptations that enhance its stability/activity with peroxide [123].

Electrocatalysis of P450s (i.e. providing electrons directly from an electrode) provides a further means by which P450 turnover can be induced in the absence of protein partners and nicotinamide coenzymes. As with the peroxide shunt, there are several drawbacks relating to such phenomena as (a) denaturation/ unfolding of the P450 at an electrode surface, (b) problems associated with providing temporally distinct electrons (enabling binding of dioxygen to ferrous heme iron prior to further reduction), and (c) turnover of P450 in absence of substrate, leading to production of damaging radicals. Notwithstanding these problems, there are examples of successful exploitation of electrocatalysis. For example, Es-tabrook and co-workers demonstrated turnover of rat CYP4A1 in a system that used the mediator cobalt sepulchrate to transfer electrons between an electrode and the dissolved P450 and rat CPR, or a rat CYP4A1-CPR fusion protein. Rates of formation of the natural hydroxylated fatty acid product (12-hydroxydodecanoic acid) were comparable with those achieved using NADPH as the electron donor [124]. Direct electrochemistry of immobilized human CYP2E1 has been demonstrated, and recent studies also reported reduction of P450BM-3 immobilized on a graphite electrode [125, 126].

Given problems with artificial routes to driving P450 catalysis, the cost implications relating to use of a coenzyme driven system may not be an overriding factor mitigating against exploiting such systems for biotransformations. Indeed, when the rapid turnover rates and tight coupling of electron transfer to substrate oxygenation demonstrated by the P450BM-3 (and P450cam) systems are considered, the operation of a "traditional" P450 turnover system may have distinct advantages over the aforementioned electrocatalytic and peroxide shunt systems. The expense of the coenzyme NADPH can be substantially offset by inclusion of a NADPH-regenerating system (e.g. glucose 6-phosphate dehydrogenase) in which NADP+ is reduced back to NADPH at the expense of exhausting a much cheaper chemical (glucose 6-phosphate). A similar system can be used to regenerate NADH from NAD+ (e.g. glucose dehydrogenase and glucose). Protein engineering has also been used to make substantial changes in coenzyme specificity from NADPH towards the much cheaper NADH for both mammalian CPR and the BM-3 enzyme, with replacement of a FAD-shielding aromatic residue in both enzymes (W676A in human CPR and W1046A in P450BM-3) effecting huge switches in catalytic efficiency (kcat /Km) towards NADH (~1000-fold and -5700fold, respectively) [59, 127].

Another exciting development in the P450 field has been the identification of a P450 system which uses NAD(P)H directly without requirement for any redox partners. The Fusarium oxysporum P450nor (CYP55A1) binds NADH directly to a P450 and uses electrons from the coenzyme to reduce two molecules of NO to N2O [128] (Fig. 5.3e). Structural studies are providing important insights into mechanistic aspects of this reaction, although it is not immediately obvious whether such a reductive system could be evolved into one capable of supporting P450 oxygenase chemistry [129].

Heal Yourself With Qi Gong

Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

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