A Fluorescent Readout for the Oxidation State of Electron Transporting

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A fluorescent readout for the oxidation state of electron transporting proteins in cell free settings Sergii Pochekailov, Rebecca Ruth Black, Venkata Pramod Chavali, Arjun Khakhar, and Georg Seelig ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00274 • Publication Date (Web): 06 Apr 2016 Downloaded from http://pubs.acs.org on April 10, 2016

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ACS Synthetic Biology

A fluorescent readout for the oxidation state of electron transporting proteins in cell free settings Sergii Pochekailov,∗,† Rebecca R. Black,† Venkata Pramod Chavali,† Arjun Khakhar,† and Georg Seelig∗,†,‡ Department of Electrical Engineering, University of Washington, Seattle, 98195, WA, USA, and Department of Computer Science and Engineering, University of Washington, Seattle, 98195, WA, USA E-mail: [email protected]; [email protected]

Abstract

that fluorescence of a direct fusion between P450 and GFP was sensitive to P450 activity, suggesting that our approach is applicable to an even broader class of proteins, which undergo a redox state change during their work cycle.

Pathways involving sequential electron transfer between multiple proteins are ubiquitous in nature. Here, we demonstrate a new class of fluorescent protein-based reporters for monitoring electron transport through such multi-stage cascades, specifically those involving ferredoxin-like electron transporters. We created protein fusions between mammalian Adrenodoxin (Adx) and plant Ferredoxin (Fdx) with fluorescent proteins of different colors and found that the fluorescence of such fusions is highly sensitive to the redox state of the electron transporter. The increase in fluorescence from the oxidized to the reduced state was inversely proportional to the linker length between the fusion partners. We first used our approach to quantitatively characterize electron transfer from NADPH through Adrenodoxin Reductase (AdR) to Adrenodoxin (Adx). Our data allowed us to build a detailed mathematical model of this mitochondrial electron transfer chain and validate previously proposed mechanisms. Then, we showed that an Adx-GFP fusion could serve a sensor for the activity of bacterial Type I Cytochrome P450s (CYPs), a very large class of enzymes with important roles in biotechnology. We further showed

Keywords Cytochrome P450, fluorescent protein, enzyme activity sensing, GFP sensor

1 Introduction Ferredoxins are a family of small, soluble (1, 2) iron-sulfur (2–5) proteins, which are found across all domains of life (2, 3, 5–8). Their main biological role is to act as electron transporters in multi-stage electron transfer chains (2, 3, 5, 9, 10) (Fig. 1 and Table 1). For example, during photosynthesis plant ferrodoxin transfers electrons from photosynthetic complex I to the enzyme ferredoxin:NADPH reductase. Bacterial ferredoxin analogues such as putidaredoxin are electron donors to bacterial CYP that catalyzes the oxidation of wide variety of biological substrates. Adrenodoxin, a human analogue of ferredoxin acts as an electron donor for several mitochondrial CYPs involved in steroid biosynthesis and other processes (reviewed in (11–15)). Electron transfer chains involving ferredoxin and its analogues typically consist of three main

∗ To

whom correspondence should be addressed of Electrical Engineering, University of Washington, Seattle, 98195, WA, USA ‡ Department of Computer Science and Engineering, University of Washington, Seattle, 98195, WA, USA † Department

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components. A reductase enzyme that acquires electrons by oxidizing a small molecule “fuel” such as NADPH; the ferredoxin — an electron transporter that accepts electrons from the reductase; and an electron acceptor that is reduced by the transporter and uses the electron to perform work (16, 17). However, although this general architecture is well established, a vast majority of biological “three-component” electron transfer chains remain uncharacterized. There are over one thousand members of class I CYPs in bacteria (18) but often their cognate electron transport partners are not known, much less the details of the electron transfer mechanism. A better understanding of ferredoxin-based electron transport requires easyto-use tools for reading out electron flow through such transfer chains. Here, we present a broadly applicable GFPbased assay for monitoring charge of ferredoxins and of other iron-containing enzymes in real time. We hypothesized that fluorescence quenching should be observable in protein fusions between GFP and iron-containing enzymes such as ferredoxins or cytochromes P450. We further conjectured that the degree of quenching should depend on the redox state of the protein fused to GFP. Our approach is motivated by the observations that purified GFP can form a donor-acceptor complex with certain organic electron acceptors (19) or electron accepting proteins (20) and, moreover, that GFP exhibits a red shift of fluorescence in the prescence of electron acceptors such as cytochrome c, flavin mononucleotide or oxidized nicotinamide adenine dinucleotide (NAD+ ) (21). We first tested our hypothesis using Adx as the electron transporter and found that a GFP-Adx fusion protein is indeed highly sensitive to the Adx oxidation state. Below, we systematically characterize this effect by varying parameters such as the concentrations of various redox pathway components, the length of the linker between GFP and Adx and the type of fluorescent protein. We will further show that similar quenching effects can also be observed with other ferredoxins and even in direct fusions between CYPs and GFP. We demonstrate the utility of our sensors in two different applications. First, we use our extensive experimental data to screen a family of 25 models for electron transfer from NADPH through

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AdR to Adx and, eventually, atmospheric oxygen. Each model represents a different hypothesis for the type and order of protein-protein interactions occurring during electron transfer. By analyzing how well different potential models fit the data we test various hypotheses reported in the literature about the reaction network architecture for this system (30–33). Second, we will show that GFP-Adx can serve as a sensor for characterizing the activity of wide range of CYPs. For this demonstration, we build on previous work showing that Adx can serve as a nonphysiological redox partner for many P450s (5), We will use two different bacterial cytochromes, CYP106A2 and CYP264A1 to show that Adx-GFP fluorescence is sensitive to both enzyme concentration and activity. Our results demonstrate a general and broadly applicable fluorescence-based platform for characterizing CYP activity, removing a major hurdle for the high-throughput functional screening of currently uncharacterized CYPs.

2 Results 2.1 GFP fluorescence depends on the Adx redox state We begin by demonstrating that GFP fluorescence is sensitive to the Adx oxidation state when Adx and GFP are colocalized. Purified Adx, while exposed to air, exists in an oxidized state (34), due to oxidation by atmospheric oxygen. Adx can be reduced by its native redox partner AdR which in turn obtain electrons by NADPH oxidation. Alternatively, Adx can be reduced by artificial electron donors such as sodium dithionite (NaDTT, or Na2 S2 O4 ). During transfer of electrons from an electron donor to an electron acceptor, Adx changes the oxidation state of its ironsulfur cluster, from Fe2+ to Fe3+ and back. Fe3+ ions are known to be stronger electron acceptors than Fe2+ (35, 36), and we thus expected fluorescence quenching in an GFP-Adx fusion protein to be stronger for Fe3+ Adx and weaker for of Fe2+ Adx. To test our hypothesis, we prepared buffer solutions with GFP-Adx and NADPH to which we added AdR after an initial equilibration period. A


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truncated version of Adx was used for all experiments, because it can donate electrons to bacterial P450s better than the wild-type version (23, 33). While wild-type Adx consist of 128 amino acids, the truncated version has only 4–108 residues. In addition to GFP fluorescence we monitored absorbance at 340 nm, which is characteristic for reduced NADPH and thus provides a direct readout of NADPH concentration. GFP fluorescence sharply increased upon addition of AdR, consistent with the hypothesis that Fe3+ in oxidized Adx is a stronger quencher than Fe2+ in reduced Adx (Fig. 2A(ii); for detailed description of our data processing method see supplementary Figs. S1 and S2). Fluorescence then slowly and gradually decreased for an extended time period before sharply dropping to a lower level consistent with oxidized GFP-Adx. Absorbance at 340nm linearly decreased over the course of the experiments, indicating gradual depletion of NADPH. The sharp drop observed for the GFP fluorescence coincided with the moment when all NADPH was used up and absorbance reached its minimum value. In a negative control experiment we added buffer instead of AdR and only observed a slight decrease of fluorescence and absorbance due to dilution. We note that in all of our experiments, we observed a gradual, approximately linear increase in background fluorescence. In the figures, all the data are shown after subtraction of the background measured in the control sample without AdR. Our data suggest that under the conditions of the experiment the electron flow from NADPH to Adx is faster than the subsequent oxidation of Adx by atmospheric oxygen, and hence Adx stays reduced as long as there is NADPH present in solution. We refer to the period for which the fluorescence of GFP is unquenched as the ”reduced state time” and use this parameter as an indicator of overall activity of the reaction components in the experiments below. To confirm that the fluorescence increase is in fact the result of electron transfer between AdR and Adx rather than being caused by a non-specific interaction between AdR and GFP, we also performed an experiment using Na2 S2 O4 which can supply electrons directly to Adx, without the need for protein co-factors. Again, we observed an initial increase in fluorescence when Na2 S2 O4 was

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added at concentrations of 3 mM or higher, followed by a return to the quenched state after all available electron donors were oxidized (Fig. S4). In an additional control experiment, we asked whether a stoichiometric mixture of GFP and Adx would exhibit similar behavior to an GFP-Adx fusion but we observed no fluorescence change when AdR was added, suggesting that close colocalization of Adx and GFP is necessary for efficient quenching (Fig. S5). To confirm this result, we created a GFP-Adx fusion protein with a linker containing a TEV protease recognition site. As expected, fluorescence gradually increased in a reaction with TEV as the protease cleaved the linker and separated GFP from Adx. We confirmed the cleavage reaction with protein gel electrophoresis (Fig. S6).

2.2 Reduced state time depends on NADPH and Adx concentrations Next, we systematically characterized the influence of NADPH, Adx and AdR concentrations on the reduced state time. We first varied the concentration of NADPH and found the reduced state time to be near-linearly dependent on the concentration of NADPH (Fig. 2B). This result is consistent with a model where reduction of Adx by AdR is more rapid than the oxidation of Adx by atmospheric oxygen. Thus, Adx effectively remains reduced as long as NADPH is available to provide electrons to AdR. When we added varying amounts of native Adx to the reaction mix while maintaining the GFPAdx concentration constant, we observed, that the high fluorescence state was shorter-lived for higher Adx concentrations. Moreover, we found that the reduced state time asymptotically approached a threshold value (Fig. 2C). We reasoned that additional Adx increases the rate of interaction with oxygen, generating additional demand for electrons from AdR. This increased demand results in increased NADPH usage and a shorter reduced state time. We found that the observed unquenching effect depends on the amount of AdR in a near steplike fashion. AdR concentration below a threshold value could not support GFP-Adx reduction, and no change of fluorescence was observed in


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rectly to Adx without any linkers (“0aa” in Fig. 3A(i)) and a fusion with a 5-amino acid truncation at the GFP C-terminus (“-5aa” at Fig. 3A(i)). We choose flexible chemically inert linkers which should not form secondary structure (37), and so, has less chance to disrupt folding of a fluorescence protein or ferredoxin (Tab. S6). We expected that closer proximity of GFP to Adx would result in a stronger effect of the Adx charge on GFP fluorescence, and thus better quenching. Indeed, upon addition of AdR we observed the biggest fluorescence increase for the -5aa fusion (21 % change) and the smallest increase for the longest fusion (10 % change) (Fig. 3A(ii)). Overall, the percentage change depended approximately linearly on the distance between GFP and Adx (Fig. 3A(iii)), consistent with an electronic quenching effect.

that case. However, once that threshold was exceeded fluorescence reached a maximal level that remained unchanged even when the AdR concentration was further increased (Fig. S7).

2.3 Fluorescence unquenching is observed in fusions of GFP with plant ferredoxin Finally, we have investigated whether the unquenching effect discovered for a GFP-Adx fusion can also be observed for other ferredoxins. Using NaDTT, we confirmed, that some other ferredoxins, in particular plant ferredoxins, show similar behavior as Adx (Fig. S8). Thus, the reported sensing mechanism is not limited to pathways involving Adx, but can also be applied for the pathways using other ferredoxins.

2.5 Other fluorescent proteins also can be reversibly unquenched

2.4 Stronger quenching is observed with shorter linkers.

To investigate whether redox state dependent fluorescence quenching could be observed with fluorescent proteins other than GFP, we created fusions of Adx with blue and cyan fluorescent proteins (BFP and CFP, respectively), as well as with mOrange and mCherry(Fig. 3B). In each case we created both N- and C-terminal fusions. BFP and CFP are mutants of GFP derived from Aequorea victoria (38), while mOrange and mCherry are similar to a red fluorescent protein (RFP) derived from Discosoma coral (39). While mutants belonging to the same family differ only by few amino acids, there is very little sequence similarity between GFP and RFP derivatives. We observed some degree of reversible unquenching for all tested fusions suggesting that this effect is not sequence specific. Rather, any fluorescent protein can be quenched by a properly colocated electron accepting protein. The magnitude of fluorescent change, however, strongly depended on the protein. Bigger changes were observed for GFP-derived proteins, while RFP-derived ones exhibited smaller changes. Specifically, we observed the most pronounced changes in fluorescence for GFP, followed by BFP, CFP, mOrange and mCherry. Interestingly, mOrange C-terminus fusions were more sensitive to quenching than Nterminal fusions, while for GFP, BFP, and CFP the

We hypothesized that the effect of reverse GFP unquenching is of electronic nature, i.e, that it is caused by the influence of positively charged iron atom on the GFP fluorophore. If this were true, the magnitude of the fluorescence change should be inversely dependent on the distance between the fluorophore and the Fe2 S2 cluster. Therefore, the length of a linker connecting GFP with Adx should matter: the shorter the linker, the more pronounced the effect. Moreover, different degrees of quenching might be expected in N- and C-terminal fusions between GFP and Adx, because the distance between Fe2 S2 cluster and fluorophore is likely different for different fusions. We first compared N-terminal (GFP-Adx) and C-terminal (Adx-GFP) fusions of GFP to Adx and found that fluorescence changes were negligible for Adx-GFP (Fig. S5). Therefore, we used Nterminus GFP-Adx fusion for all further experiments. In order to understand how the fluorescence change depends on the distance between GFP and Adx, we created GFP-Adx fusions with linkers containing 15, 10, or 5 amino acids (“15aa,” “10aa,” and “5aa,” in Fig. 3A(i)). Additionally, we created a fusion in which GFP was fused di-


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opposite was true. mCherry fluorescence changes were very small for both orientations. To investigate whether the observed fluorescence changes are homogeneous over the entire excitation spectrum, or specific to certain wavelengths, we measured the entire excitation spectra of fusions between different fluorescent proteins and Adx (FP-Adx) after addition of AdR (Fig. S9). We found that the most pronounced, reversible spectral changes occurred at the highest excitation peak (Fig. S10). Importantly, these peaks do not overlap for different fluorescent proteins such that multiple reporter fusions can be used simultaneously.

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errors between the predictions and the data, allowing the kinetic parameters to be varied. Fits to the best performing model (model 1) are shown in Fig. 2 as thin black lines. By comparing the performance of different models, we can draw several conclusions: First, the models proposed by Ziegler et al. (Model 2, Fig. S11) and Behlke et al (33) (Model 5, Fig. S11) both resulted in good fits to our data. Second, four of the five best models include the formation of a multi-protein complex between one AdR and two Adx. However, unlike the mechanism proposed by Behlke et al.(33), none of those models include an initial Adx dimerization step. This is not a surprise, as we here used a truncated version of Adx which unlike full-length Adx does not form dimers. Indeed we found that including a Adx dimerization step preceding the formation of the AdR · 2 Adx complex did not improve model quality. Third, oxidation of reduced Adx by atmospheric oxygen is rate limiting or close to rate limiting with rate constants ranging between 0.391.27 min−1 for the 10 best models. This value agrees well with a previously reported value of 0.13 min−1 (16). The slightly faster rate found in our experiments is likely due to the fact that our approach resolves intermediate reaction steps while Hanukoglu et al. measure an aggregate reaction. Fourth, Adx has two charge centers and could in principle, like AdR, carry two electrons simultaneously. However, any model assuming that Adx is transporting two electrons at a time resulted in a poor fit to the experimental data (models 19, 20, 23). A few caveats remain. Most importantly, our data did not allow us to conclusively discriminate between the best performing models, primarily because the time resolution was not sufficient to capture the rapid initial reduction of AdR and Adx. Moreover, sensing the Adx electron transfer chain involves using an Adx-GFP sensor which may interact with its redox partners in a manner that could be distinct from the native Adx. For modeling convenience, we lumped the two species together into a single Adx species, an assumption that only holds if the reaction rates do not differ significantly or if the electron transfer between the two species is sufficiently fast. Finally, as explained above, we subtracted a gradually increas-

2.6 A model for the reactions of electron transport by Adx The Adx-GFP sensor described above made it possible to measure electron flow through the NADPH-AdR-Adx electron transfer chain for a wide range of experimental conditions (25 traces total). Here, we ask whether our data is compatible with previously suggested mechanistic models for this pathway and to what extent our measurements allow us to distinguish between competing hypotheses about the reaction network architecture. Prior work on the adrenodoxin pathway has identified features of the reaction mechanism and has led to several proposals for the reaction pathway. Lambeth et al. showed that oxidized Adx and AdR form slowly dissociating complexes with 1:1 stoichiometry (30). Muller et al. studied the crystal structures of AdR and Adx and argued that these structures suggested potential electron transfer paths between an AdR and Adx in complex (31). Ziegler et al. used structural data to argue for a reaction mechanism involving a two stage transfer of electrons, where two Adx molecules sequentially interact with a reduced AdR (32). Behlke et al. proposed a model in which Adx dimers bind to reduced AdR and the two Adx subsequently get reduced and released one by one (33). Here, we mathematically formalized these models and also created a number of variants corresponding to alternative hypotheses. In total, we fit 25 models to the experimental data and ranked them based on the minimum summed mean square


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ing background signal from the data and this background subtraction somewhat increased the variability in the data. Still, we believe that the approach developed here can help to uncover the mechanism underlying electron transfer in multistate cascades.

We independently confirmed the activity of CYP106A2 using a steroid fluorescence assay, that takes advantage of the observation that some hydroxylated steroids can form fluorescent products under strongly oxidative, acidic conditions(40, 41) (Supplementary results and Fig. S12). The same assay allowed us to demonstrate that a GFP-Adx fusion is able to support electron transport from AdR to CYP106A2 (Fig. S12). Finally, we asked whether the main drug metabolizing human CYPs, CYP2C9 and CYP3A4, can interact with an GFP-Adx, and thus, whether it is possible to build a drug sensor using our approach. However, we did not observe a decrease of the reduced state time in these experiments, suggesting that there is no effective electron transfer between Adx and CYP2C9 or CYP3A4. We even observed that the reduced state time increased for reaction mixtures where those CYPs were present (Fig. S13), possibly because CYP2C9 and CYP3A4 are shielding Adx from the influence of oxygen, thus preventing oxidation. (Samples with deactivated CYP showed similar reduced state times as the sample without CYPs, which suggests that the CYP is in fact responsible for the observed reduced state time increase.) The lack of electron transfer between GFP-Adx and these human CYPs is not necessarily surprising, given that in their native pathway these enzymes accept electrons directly from an NADPH-P450 reductase without the need for iron-containing Adx-like electron transport protein.

2.7 Duration of unquenching is sensitive to CYP enzyme and substrate concentrations Adx can donate electrons to a number of mammalian and bacterial CYPs, and adding a CYP to the NADPH-Adx-AdR electron transfer chain should thus increase the rate of electron flow. This increased flow should be observable as a decrease of the reduced state time of a GFP-Adx fusion. Moreover, this effect should be sensitive to the enzyme activity and thus, at least over some concentration range, to the substrate concentration. To demonstrate that reduced state time is sensitive to CYP activity we chose two bacterial CYPs, CYP106A2 and CYP264A1 that were both previously shown to accept electrons from Adx (10, 23). We used 21-hydroxyprogesterone as a substrate for CYP106A2, and 4-methyl-3-phenylcoumarin as a substrate for CYP264A1. As expected, addition of CYP106A2 to the electron transfer chain led to an accelerated return to the quenched state, with higher enzyme concentrations resulting in a more rapid fluorescence drop (Fig. 4A). Furthermore, increasing the concentration of 21-hydroxyprogesterone also resulted in faster consumption of NADPH. However above a threshold value of 200 µ M, NADPH consumption became independent of the substrate concentration (Fig. 4B), likely because at such high concentrations the enzyme is already working at full capacity, and additional substrate cannot increase the reaction rate. Addition of CYP264A1 also resulted in a shorter reduced state time (Fig. 4C). The size of the shift in the reduced state time was the same in the presence or absence of substrate. We speculate, that the turnover of CYP264A1 in this experiment is dominated by non-specific catalysis of buffer components (remains of imidazole which was used during proteins purification) rather than the specific hydroxylation of the substrate.

2.8 GFP-P450 fusion proteins exhibit redox state dependent fluorescent changes Similarly to Adx, CYP is a heme iron containing protein that changes its redox state during the catalytic cycle. We thus set out to investigate whether CYP can influence GFP fluorescence in the same way as Adx does. We created CYP106A2-GFP fusion proteins and observed fluorescence quenching for both N and C terminal fusions of CYP to GFP (Fig. 5A). This quenching was partially relieved when electrons were supplied to the CYP (Fig. 5B and suppl. Fig. S3). The reduced state time was linearly dependent on NADPH concen-


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natives. The main difference between the models by Ziegler et al. (32) and Behlke et al. (33) is that the former assumes that two Adx molecules interact sequentially with AdR while the latter assumes that 2 Adx bind to AdR as a dimer before getting reduced a and released. Given that the initial reduction of AdR and Adx occurs on a timescale that could not be resolved with our experimental setup, we were unable to identify the precise ordering of complex formation and redox reactions. However, in principle this issue can be addressed using stopped flow kinetics measurements (45) and the method developed here can be used to further refine mechanistic models of electron transfer chains. We also showed that Adx-GFP can serve as a sensor for the activity of bacterial CYPs. The reduced state time was inversely proportional to the amount of CYP added to the reaction and was also sensitive to substrate concentration at least in the range below 100 µ M. Given that Adx can donate electrons to a wide range of non-native partners, our sensor technology forms an easy-to-use assay for characterizing the enzymatic activity of CYPs with unknown native redox partners. An intriguing but also challenging future application of our assay is the detection of CYP activity in vivo. A live cell sensor would be particularly intriguing in the context of directed evolution experiments geared towards creating new enzymes or improving the effectiveness of existing enzymes for biosynthesis (23, 46–48), sensors (49) and bioremediation (50) and other biotechnology applications. The major obstacle preventing a direct application of our approach to in vivo measurements is the fact that living organisms maintain a steady supply of NADPH, making it impossible to perform reduced state time measurements that rely on fluorescence changes occurring upon NADPH depletion. Still, we believe that it is possible to overcome this obstacle. For example, if the reducing agent AdR is put under control of an inducible promoter, we can measure the size of the fluorescence increase upon induction of AdR. In conclusion, we here presented a novel approach to sensing electron transport through multistage electron transfer chains that can be broadly useful for quantitatively characterizing the activity of CYPs and other proteins that change electronic

tration, similarly to what we observed for a GFPAdx fusion (Fig. 5C). However, the observed absolute fluorescence changes were considerably smaller than those measured for GFP-Adx fusions (around 3% rather than 21 %). These smaller changes are likely due to a less favorable configuration of the CYP charge center relative to the GFP fluorophore, possibly a result of the large size of CYP106A2 compared to Adx (protein size comparison is in Table S4). Still, this result suggests that direct CYP-GFP fusions can be used to report on CYP activity in real time. This is especially useful for CYPs that cannot accept electrons from Adx or similar non-native transporters.

3 Discussion In this paper, we described a novel assay for monitoring the redox state of electron transporting proteins in vitro. We found that the fluorescence of a fusion between an electron transporting protein and a fluorescent protein can be sensitive to the oxidation state of the former. For example, the fluorescence of an Adx-GFP fusion was found to be 21% higher when Adx was reduced. The approach is compatible with a variety of electron transporters and fluorescent proteins; fusions with different color fluorescent proteins could thus be used to simultaneously characterize multiple reaction stages in more complex systems, such as photosynthesis pathways (27, 28) or pathways involving several CYPs (42). Our approach makes it possible to directly observe the redox state of a protein of interest rather than using NADPH consumption as a proxy for protein activity (43, 44). However, like an NADPH consumption assay, our approach is compatible with a simple plate reader making it possible to screen a large number of reaction conditions simultaneously. We used the quantitative fluorescence kinetics data collected with our sensor technology to compare and evaluate a set of 25 models each corresponding to a different reaction mechanism for electron transfer from NADPH to AdR and Adx. We found that our data was quantitatively and qualitatively compatible with two previously proposed mechanisms and could discriminate them from less biochemically realistic alter-


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4.3 GFP-Adx reduction with sodium dithionite

4.5 GFP-Adx fusion cleavage with TEV protease

Measurements of GFP-Adx reduction with artificial electron donor sodium ditionite (Na2 S2 O4 ) were broadly similar to other kinetic experiments. Experiments were started with 4 µ M solution of GFP-Adx fusion (or GFP as a control) in 0.1 M TRIS-HCl buffer with pH 7.5. The reduction reaction was initiated by adding Na2 S2 O4 solution to a final concentration ranging from 100 to 20000 µ M. The raw data were processed to remove the effect of direct interaction between GFP and Na2 S2 O4 . To do that, we measured kinetics of GFP-Na2 S2 O4 interaction and fit the resulted curve with exponential function y = a × ek×t + c, where t is the kinetic time variable, y is the GFP fluorescence and a, c, k are coefficients. After finding the coefficients, we subtracted resulted curve from GFP-Adx kinetic to obtain the presented plot (Fig. S4).

Cleavage of the GFP-Adx fusion with TEV protease was performed in 0.1 M TRIS-HCl buffer pH 7.5. The concentration of GFP-Adx fusion was 4 µ M. After initial measurement of GFP-Adx fusion fluorescence, TEV protease (Life Technologies, Novex AcTEV Protease, cat. no. 12575–015) was added to final concentration 10 uM. Kinetics were measured overnight, and subsequently a protein gel electrophoresis experiment was performed. Acrylamide gel for protein separation was prepared according to (55); we used 4% stacking layer and 12% resolving layer; gel thickness was 1 mm, 15 wells. To each well, we loaded 20 uL of reaction mixture, or 20 units of TEV protease, or 2 uL of purified proteins as controls (concentrations of purified proteins was in range of 300– 900 uM). All proteins were heated at 98◦ C for 5 minutes prior to loading. Gel electrophoresis was performed for 1 hour at 120 V. After that, gel was washed with distilled water 3 times, and microwaved for 75 seconds at maximum power with GelCode Blue Stain Reagent (until reagent start boiling). The stain was removed, water was added and gel was microwaved again at maximum power for 75 seconds. After that, the gel was imaged and the image was processed in ImageJ software to increase contrast and to remove background.

4.4 Steroid Fluorescence Assay In 1.7 mL eppendorf tubes, we prepare a protein mixture using measurement buffer (Table S2) as a solvent. We add there AdR, Adx (or GFP-Adx fusion, or both) and CYP106A2 in molar proportion 3:20:10, so the total volume of solution become 0.15 mL. After that, we started the reaction by adding 0.1 mL of a reaction mixture, which contained NADPH, glucose6-phosphate, glucose-6-phosphate dehydrogenase and 21-hydroxyprogesterone. The concentration in the final reaction mixure were as follows: NADPH 200 µ M, glucose-6-phosphate 2.5 mM, 15 units of glucose-6-phosphate dehydrogenase and 21-hydroxyprogesterone 0.5 mM. After some time, we extracted steroids by adding 0.25 mL of 4-methyl-2-pentanone and waiting for 30 minutes, vortexing the mixture several times. Water and organic layers were then separated by centrifuging the resulted emulsion at 14000 RPM for 10 minutes at room temperature. The organic layer was transferred to 96 wells plate, made of polypropylene. A mixture of dry concentrated sulfuric and acetic acid was added to the extracted steroid solution and left to react for 1.5 hours. After that, fluorescence was measured using plate reader.

Acknowledgement This work was supported by ONR award N00014-13-1-0880 and the University of Washington Royalty Research Fund. We thank Rita Bernhardt and Frank Hannemann for their generous gift of CYP264A1 enzyme. We also thank Bill Atkins, Caleb Woods and Michelle Redhair for their genersous gift of CYP2C9 and CYP3A4 proteins. We are grateful to Peter F. Guengerich and Irina Pikuleva for plasmids containing AdR and Adx genes. We thank Eric Klavins and members of the Seelig lab for helpful comments on this manuscript. Supporting Information Available: Enhanced description of methods mentioned in the text; figures describing kinetic data processing, figures describing interactions of GFP-Adx fusions with Na2 S2 O4 ; figure showing negative controls for GFP-Adx reversible quencing; figures showing


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AdR titration; figures and text explaining GFP fused with different FPs, spectroscopy investigations of FP-Adx fusions; modelling behavior of systems containing GFP-Adx fusions; section describing CYP106A2 steroid oxidation assay; figures showing interaction of GFP-Adx fusion with mammalian P450s; list of plasmids used in this work; Tables containing information about E.coli growth media; buffers; protein purification yields; protein sizes and their molecular weights; linkers between GFP and Adx. This material is available free of charge via the Internet at http: //pubs.acs.org/.

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A Fluorescent Readout for the Oxidation State of Electron Transporting

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