Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis

Joseph B. Lim,1 Kimberly A. Barker,1 Kristen A. Eller,1 Linda Jiang,1 Veronica Molina,2 Jessica F. Saifee,3 and Hadley D. Sikes1* 1

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

2

Department of Chemical Engineering, Polytechnic University of Puerto Rico, San Juan 00918, Puerto Rico Neuroscience Program, Wellesley College, Massachusetts 02481

3

Received 4 February 2015; Accepted 23 August 2015 DOI: 10.1002/pro.2793 Published online 27 August 2015 proteinscience.org

Abstract: As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain’s function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we Abbreviations: P450 BM3, cytochrome P450 monooxygenase from B. megaterium; P450 cam, cytochrome P450 monooxygenase from P. putida; WT, wild type; kcat, R-H, turnover rate constant with respect to hydroxylation; Km, R-H, Michaelis constant with respect to hydroxylation; kcat, leakage, turnover rate constant with respect to leakage; Km, leakage, Michaelis constant with respect to leakage; Kd, dissociation equilibrium constant; kon, association rate constant; koff, dissociation rate constant; T50, incubation temperature at which half of protein becomes denatured after 10 min; NADPH, nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; H2O2, hydrogen peroxide; O2 2 , superoxide; IPTG, isopropyl b-d-1-thiogalactopyranoside; KPi, potassium phosphate; HRP, horseradish peroxidase; ABTS, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); CO, carbon monoxide; Tris, tris(hydroxyl)aminomethane; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis Additional Supporting Information may be found in the online version of this article. Grant sponsor: National Science Foundation Graduate Research Fellowship; Grant sponsor: Burroughs Wellcome Fund Career Award at the Scientific Interface; Grant sponsor: Massachusetts Institute of Technology. *Correspondence to: Hadley D. Sikes, Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: [email protected]

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investigated were far more deleterious than other mutations that have been used to change substrate specificity. Keywords: cytochrome P450; site-directed mutagenesis; leakage; reactive oxygen species; hydrogen peroxide; spin state; thermostability

Introduction Cytochromes P450 (P450s)1 are a family of heme containing redox enzymes2–4 that perform a variety of different biological functions in a diverse range of organisms.5–7 In a typical reaction cycle of a subset of P450s, electrons from nicotinamide adenine dinucleotide phosphate (NADPH) are shuttled through reductase domains to the oxidase domain, where dioxygen is reduced and activated for hydroxylation of the native substrate.8 This requires exquisite coupling of activities in distinct domains. A convenient model for studying this coupling phenomenon is cytochrome P450 BM3, which fuses reductase and oxidase domains and exhibits the highest activity of any P450.9 Mutations have been introduced into this particular member of the P450 family to improve its activity towards non-native substrates10–14 and also better understand the roles of various residues.15–19 The introduction of mutations often increases the frequency of leakage20 and decreases coupling efficiency.11 It is important to make clear the distinction between coupling and leakage: coupling is formally defined here as the percentage of NADPH used for hydroxylation of substrate (R-H), while leakage is defined as NADPH oxidation activity (in units of nmol NADPH nmol P45021 min21, or simply min21) in the absence of substrate bound to the active site for hydroxylation (Fig. 1). While hydroxylation results in hydroxylated product (R-OH) and water, leakage does not produce hydroxylated product, since it is performed without bound R-H, and can result in either water or the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O2 2 ). It is also important to note that uncoupled hydroxylation with substrate bound can also result in the production of water or ROS with bound R-H not being hydroxylated and thus remaining unchanged; this, however, is distinct from leakage, since leakage occurs without R-H while uncoupled hydroxylation occurs with R-H bound. All of these reactive pathways exist, and whether each occurs depends on how the kinetics of each compare. Thus, leakage is always in kinetic competition with the hydroxylation reaction, and for some combinations of reactant concentrations and kinetic parameters, can divert electrons away from hydroxylation of target substrate, and thus decrease coupling. Although leakage is undesirable in applications requiring hydroxylation, its production of ROS may prove useful in other contexts. Our lab recently

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showed, for instance, that P450 BM3 can be engineered in a whole cell screen for enhanced H2O2 production by coexpressing the enzyme with HyPer, a fluorescent sensor specific to peroxide.21 Enzyme variants with enhanced leakage and H2O2 production could be used as novel reagents in quantitative studies of how H2O2 impacts cell fate. Methodologies for producing peroxide in quantitative studies of redox biology have been increasingly shifting from bolus addition to more continuous, low-level endogenous generation using soluble, genetically encoded enzymes, which may better reflect physiological conditions.22–24 Leakage thus has consequences not only for hydroxylation but also for applications in which ROS production is desired. In addition to its relevance to practical engineering applications, leakage also holds interest as it relates to a fundamental understanding of P450 biocatalysis. A substrate for P450 BM3 to hydroxylate has historically been considered necessary to trigger the catalytic oxidation of NADPH because the binding of target substrate to the enzyme is thought to induce required changes in conformation, reduction potential, and spin state that make the electron transfers favorable.25 Leakage is catalytic oxidation of NADPH without bound R-H substrate, raising the question of whether leakage-enhancing mutations induce similar protein property changes thought to be required for favorable electron transfer. While leakage in P450 cam, another member of the P450 family, has been extensively studied,20 leakage in P450 BM3 is relatively unexplored. Although many studies often note the coupling of P450 BM3 in hydroxylation, only a few have noted mutations, such as A82W,19 T268N/F393H,18 and I401P,26 that significantly increase the rates of leakage activity. The reaction conditions in these studies were not identical, complicating comparison of these variants. These mutations were also associated with different property changes without bound substrate, including structural conformation, reduction potential, and spin state content of the heme domain. Systematic and quantitative investigation of these mutations should increase knowledge of which mechanistic properties are important with respect to leakage in P450 BM3’s catalytic cycle. In this work, we asked the following: do combinations of individual leakage-enhancing mutations increase leakage even further? Are the properties known to be important in hydroxylation also

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Figure 1. Schematic of P450 hydroxylation and leakage. NADPH binds to the reductase domain and donates electrons to the FAD and then FMN cofactors, which then transfer the electrons to the heme in the oxidase domain. In coupled hydroxylation, O2 and R-H bind at distinct sites, and O2 is reduced and activated to hydroxylate R-H, transforming it into R-OH. In leakage, the electrons are still transferred to the heme and used to reduce O2, but this may result in water or ROS, such as H2O2 and O2 2 . R-H is not involved in the leakage reaction.

relevant in leakage? To what extent does leakage of electrons lead to ROS generation? Are any other protein properties associated with increases in leakage? To address these questions, we introduced the mutations A82W, T268N/F393H, and I401P into wild-type (WT) P450 BM3, and then combined these mutations, resulting in four new variants: A82W/I401P, A82W/T268N/F393H, T268N/F393H/I401P, and A82W/T268N/F393H/I401P (Fig. 2). We did not investigate T268N or F393H without the other since it has already been reported that only combining these two mutations together results in enhanced leakage; each of these mutations alone does not enhance leakage.18 Using this set of variants, we investigated the relationship between increased leakage and other key properties of the enzyme, such as the stability of the protein fold and electronic properties of the heme.

Modeling effects of leakage on coupling with respect to hydroxylation To explore the potential effects of increasing leakage, we modeled leakage and hydroxylation of palmitate27,28 as two competing kinetic reactions, implementing them as differential equations in Matlab (Mathworks). The parameters (Supporting Information Table S2), reaction scheme (Supporting Information Fig. S1), equations, and code are shown in the Supporting Information. Since kon and koff values have never been measured for P450 BM3 and a

Results and Discussion We first modeled leakage and hydroxylation as competing kinetic reactions to illustrate leakage’s potential impact on coupling. We then engineered previous leakage-enhancing mutations18,19,26 into P450 BM3 and combined them, determining the effects of these mutations on leak rate, spin-state content, thermostability/evolvability, and H2O2 production, and thus exploring the relationship between leakage and other key protein properties. Finally, we measured hydroxylation of a model substrate by variants with differing leakage activities.

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Figure 2. Schematic for lineage of variants with enhanced leakage derived from wild-type P450 BM3. Variants found to exhibit enhanced leakage in previous studies colored in green. Variants directly derived from variants measured in previous studies colored in blue. Variant containing all mutations in this study colored in red. The T268N variant was not generated since it was already known that only combining this mutation with the F393H mutation, rather than either of these mutations alone, increases leakage.18

Leakage in Cytochrome P450 BM3 Reaction Cycle

Figure 3. A: Time-course plots of NADPH and hydroxylated product R-OH with different kcat, leakage values, assuming kon for RH and P450 BM3 to be 105 M21 s21 and using Kd, Km, and kcat, R-H values for palmitate.27,28 Black, red, blue, and green represent kcat, leakage of WT (as measured and later reported in this study) multiplied by 108, 101, 102, and 103, respectively. Solid lines represent concentration of NADPH. Dashed lines represent concentration of R-OH. B: Coupling plotted against the different kcat, leakage values used in (A). Coupling is defined as the percentage of NADPH used for hydroxylation. Pink squares, purple circles, maroon triangles, and gray triangles represent kon values for enzyme binding to R-H of 105, 106, 107, and 108 M21 s21, respectively.

particular substrate, we used a kon range of 105 to 108 M21 s21 for association of enzyme with R-H (typical for association of enzyme with substrate29) and calculated the effects of increasing kcat, leakage (i.e., kcat measured for NADPH consumption in the absence of an R-H substrate), which resulted in higher rates of overall NADPH consumption as well as lower coupling percentages (Fig. 3 and Supporting Information Fig. S2). For illustrative purposes, Figure 3(A) assumes a value in the low end of the range of kon (105 M21 s21). Because kcat, palmitate is of the order 103, substrate hydroxylation outcompetes the leakage reaction when kcat, leakage is 10 (black dashed line), resulting in almost complete coupling of NADPH consumption and R-OH product formation. Figure 3(B) shows the impact on coupling of larger values of kon for the binding of palmitate. For larger values of kon, larger increases in kcat, leakage are required to impact coupling as severely as shown in Figure 3(A). While these simulations do not include the microscopic rate constants of the individual steps in the catalytic cycle, using kcat and Km captures the entire cycle for both leakage and hydroxylation and provides order-of-magnitude estimates of the effects of leakage on coupling. The extent to which mutations decrease coupling in P450 BM3 can vary widely. In hydroxylation of lauric acid, for example, the I401P variant exhibited a significantly higher leak rate but did not have a diminished coupling ratio compared to WT.26 Variants in the alkane hydroxylation lineage, on the other hand, showed both higher leakage and significantly lower coupling than WT with respect to P450 BM3’s native substrates.30 The results in Figure 3 show that this discrepancy in leakage’s effects on coupling may be due to differences between these variants in terms of the magnitude of kon relative to

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that of kcat, leakage. For example, if kon is set at 108 M21 s21, then leakage is generally outcompeted, as even kcat, leakage >104 min21, an amount on par with P450 BM3’s highest reported activity,9 only diminishes the coupling ratio to 78%; a kcat, leakage value on par with that typical for WT26 would decrease the coupling ratio by