The activation of C-H bonds is of significant interest given its many applications, such as conversion of methane to methanol [1], and natural product synthesis [2]. The relatively high C-H bond energy (∼80–100 kcal/mol) means that C-H activation typically requires powerful catalysts or harsh conditions that are often incongruent with the stereo- and regioselective transformations of complex, multi-functional substrates into desired products (e.g., pharmaceutical synthesis [3]). Thus, an active area of research involves finding “greener” methods of conducting such reactions [4]. In this regard, enzymatic reactions offer many advantages, including high regio- and stereoselectivity, relatively non-toxic and environmentally friendly components, and ambient reaction conditions, thus making biocatalysis attractive for myriad industrial and commercial applications [3,[5], [6], [7], [8]].

On the path to developing oxidation chemistry that satisfies the aforementioned criteria has been the cytochromes P450 (P450s). These enzymes were first described in the 1960s by Omura and Sato [[9], [10], [11]], ultimately leading to the description of thousands of P450 genes resulting in a plethora of information on P450 biochemistry, structure, and function [12]. These monoxygenases play critical roles in mammalian cellular pathways (e.g., hormone synthesis) [13], and human P450s metabolize numerous drugs [14]. While P450s perform a variety of reactions regio- and stereospecifically under physiological conditions, the hallmark reaction is hydroxylation of hydrocarbons (more precisely, insertion of an oxygen atom):R−H+NAD(P)H+H++O2→P450R−OH+NAD(P)++H2O

Following decades of work, there continues to be great interest in exploiting P450 activity [[15], [16], [17], [18], [19]]. The pharmaceutical industry has particular use for P450 systems in the preparation of drug metabolites for pharmacokinetic studies, precursor synthesis, diversification of lead compounds, and natural product synthesis [20,21]. Practical issues relevant to commercially viable P450 biocatalysis include cost, scalability, product purification, turnover number and rate, and cofactor recycling [22]. The latter challenge is tied to the catalytic mechanism and is among the most vexing given the complexity of P450 electron transfer (ET).

A thorough summary of mechanistic aspects related to P450 ET and dioxygen activation has been provided by Guengerich [23]. Fig. 1 highlights the salient aspects of the pathways relevant to this review. Starting with a six-coordinate hydrated FeIII heme-thiolate, substrate binding generates the five-coordinate heme and causes a positive shift in redox potential that initiates ET from native reductase proteins to generate FeII. Redox potentials can vary widely: P450 3A4 stabilized with nanometer-scale membrane bilayer discs in pH 7.4 phosphate buffer yielded formal potentials (vs SHE) of −220 mV for substrate-free enzyme (11 % high-spin), −210 mV with erythromycin, and − 140 mV with testosterone [24]; similar potentials for P450 BM3 (pH 7 phosphate buffer) are −368 mV and − 239 mV for substrate free and bound with arachidonic acid, respectively [25]. Such variations in redox potential coupled with concentration effects (e.g., [O2]:[P450], [reductase]:[P450]) can have significant effects on the equilibria and reaction rates in the mechanism [26,27].

In aerobic environments, FeII P450 reacts with dioxygen. Thus, the substrate-gated mechanism described above prevents the wasting of reducing equivalents as well as futile redox cycling–i.e., ET and product formation are highly coupled, otherwise under aerobic conditions without substrate, reactive oxygen species (ROS, superoxide and peroxide) would result. A second proton-coupled ET produces FeIII-OOH− (Compound 0); protonation to yield a water molecule gives rise to Compound I. Studies by Green [28], and later supported by Makris [29] and Hoffman [30] indicate that Compound I is FeIVO, with a radical cation delocalized over the porphyrin and axial thiolate ligand (i.e., P+FeIV=O). Based on a d4 electron count in tetragonal symmetry, substrate oxidation then occurs via the “rebound” reaction [31]: Compound I abstracts hydrogen from the substrate to form FeIV-OH (Compound II), which hydroxylates the substrate radical by OH• transfer [32,33].

Two additional pathways are noteworthy: (i) direct oxidation and (ii) the “peroxide shunt”. First, one can imagine oxidizing the resting FeIII-OH2 state by one- and two electrons, leading to Compounds II and I, respectively (Fig. 1) [34,35]. This opens the possibility of realizing substrate conversion through direct heme oxidation, which circumvents formation of ROS through aerobic heme reduction. A consequence of this alternative pathway may be a change in regio- and stereospecificity, as water must remain bound to the heme for the reaction to occur (cf. substrate-gated mechanism).

Second, in the peroxide shunt, reduced dioxygen is provided in the form of a peroxide which binds directly to the FeIII heme (Fig. 1). This ferric-peroxy species then leads to formation of Compound I through the native mechanism. While utilizing peroxide may present challenges such as protein degradation and low affinity of the enzyme for peroxide [36], efficient P450 peroxygenases are increasingly being discovered and characterized [[37], [38], [39]] (notably, recent work describes a P450 peroxygenase with activity on the high-value compound lignin [40]). Also, progress has been made towards engineering viable peroxygenase activity [[41], [42], [43], [44]]; this was recently exemplified by the conversion of P450 monooxygenases to peroxygenases by mutating residues in the I-helix associated with dioxygen activation [45].

Related to the catalytic mechanism are uncoupling pathways, whereby reducing equivalents are consumed without substrate oxidation. For P450, this could be benign–e.g., reduction of the ferric-peroxy species to water–or damaging, with the production of ROS as noted above. Either way, reducing equivalents are consumed, and in the case of NAD(P)H as the ultimate electron donor this can become unsustainable. An excellent review on the significance of uncoupling is provided by Hollmann [46].

Given the intricate ET involved in P450 catalysis, exploiting enzyme activity depends largely on preserving the native mechanism. This underscores the NAD(P)H requirement, which is not readily substituted. In vitro schemes that may rely on stoichiometric consumption of NAD(P)H would be prohibitively expensive. Whole-cell systems present one option, which can express all enzymatic components for native activity with good stability, and take advantage of in vivo NAD(P)H production and recycling [[47], [48], [49], [50], [51], [52], [53]]. Considerations with whole-cell systems include turnover rates, enzyme stability (in particular for heterologous expression), substrate access and product recovery, and any inhibition or toxicity related to the substrates and products. Alternatively, in vitro enzymatic cofactor recycling removes the complexity of the in vivo environment, and does not require maintaining cell cultures [[54], [55], [56], [57], [58]]. One key challenge is unwanted byproducts depending on the recycling system used (e.g., acidification with glucose-6-phosphate dehydrogenase). Protein engineering has shown some promise here; for example, rational design of a NAD+ hydrogenase to support NADPH recycling with P450 [59]. Ideally, being able to reduce the system to the core catalytic components–i.e., oxygenase, substrates–would provide a much simpler system, particularly if some components are heterogeneous (e.g., electrode). As such, one may identify the P450 “Holy Grail” as in vitro, NAD(P)H-independent catalysis: this forms the basis for the discussion below.