Structural origin of the large redox-linked reorganization in the 2

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Structural origin of the large redox-linked reorganization in the 2-oxoglutarate dependent oxygenase, TauD Christopher W. John, Robert P. Hausinger, and Denis A. Proshlyakov J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b07493 • Publication Date (Web): 01 Sep 2019 Downloaded from pubs.acs.org on September 1, 2019

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Journal of the American Chemical Society

Structural origin of the large redox-linked reorganization in the 2oxoglutarate dependent oxygenase, TauD Christopher W. John,1 Robert P. Hausinger,2,3 and Denis A. Proshlyakov1  1 2 3

[email protected] Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA

Abstract: 2-Oxoglutarate (2OG)-dependent oxygenases catalyze a wide range of chemical transformations via C-H bond activation. Prior studies raised the question of whether substrate hydroxylation by these enzymes occurs via a hydroxyl rebound or alkoxide mechanism and highlighted the need to understand the thermodynamic properties of transient intermediates. A recent spectroelectrochemical investigation of the 2OG-dependent oxygenase, taurine hydroxylase (TauD), revealed a strong link between the redox potential of the Fe(II)/Fe(III) couple and conformational changes of the enzyme. In this study, we show that the redox potential of wild-type TauD varies by 468 mV between the reduction of 2OG-Fe(III)-TauD (-272 mV) and oxidation of 2OG-Fe(II)-TauD (196 mV). We use active site variants to investigate the structural origin of the redox-linked reorganization and the contributions of the metal-bound residues to the dynamic tuning of the redox potential of TauD. Time-dependent redox titrations show that reorganization occurs as a multi-step process. Transient optical absorption and infrared spectroelectrochemistry show that substitution of any metal ligand alters the kinetics and thermodynamics of the reorganization. The H99A variant shows the largest net redox change relative to the wild type protein, suggesting that redox-coupled protonation of H99 is required for high redox potentials of the metal. The D101Q and H255Q variants also suppress the conformational change, supporting their involvement in the structural rearrangement. Similar redox-linked conformational changes are observed in another 2OG dependent oxygenase, ethylene-forming enzyme, indicating that dynamic structural flexibility and the associated thermodynamic tuning may be a common phenomenon in this family of enzymes.

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Introduction Taurine:2-oxoglutarate (2OG) dioxygenase (also termed taurine hydroxylase or TauD) is a non-heme mononuclear iron enzyme and the archetypal member of the 2OG-dependent oxygenase family.1 These enzymes typically share a 2-His-1-carboxylate iron binding motif (Fig. 1) and utilize 2OG to activate O2, generating an Fe(IV)=O (F4) intermediate that performs substrate C-H bond activation (Fig. 2).2 This reactivity allows the 2OG-dependent oxygenases to catalyze a wide range of biochemical reactions.3 TauD is found in Escherichia coli where it catalyzes the production of sulfite from taurine in sulfur-starved cells.4 Historically, TauD was proposed to utilize a hydroxyl radical rebound mechanism similar to that observed in cytochromes P450;1,5 however, recent studies have suggested that TauD and its fellow family members instead use an alkoxide-forming (AF) mechanism (Fig. 2).6,7

Figure 1. Selected residues at the TauD active site. The peptide segment proposed to be linked to structural rearrangement is highlighted in stick mode. Selected hydrogen bonding interactions (yellow) and water molecules (red) are shown. The carbon atoms of the substrates are shown in orange.8

In the AF mechanism, the F4 intermediate activates the C-H bond at C1 of taurine by hydrogen atom transfer (HAT)9 yielding a substrate radical and the Fe(III)-OH species, which rapidly transfers its proton to a nearby base and forms a vibrationally detectable Fe(III)-O– intermediate (F3). The deprotonated oxygen ligand forms the Fe-O-C bond of the alkoxo


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intermediate (FX).6,7 Subsequent cleavage of the Fe-O bond leads to the formation of sulfite and aminoacetaldehyde products.1

Figure 2. Catalytic cycle of TauD comparing the hydroxyl radical rebound (grey) and alkoxide-forming (blue) mechanisms. Vertical transitions indicate a change in the oxidation state of the Fe center. Diagonal transitions indicate a change in a protonation state. Red structures show the artificial manipulation of the enzyme used in this study to generate an in-situ model of the F3 intermediate.

This mechanism, initially proposed based on the transient Raman data for TauD6 and more recently supported by X-ray crystallography of the arginine hydroxylase VioC,7 leaves two important questions to be answered: i) the feasibility of the Fe(III)-O– intermediate under physiological conditions and ii) the identity of the base that is responsible for accepting the proton from the Fe(III)-OH group formed immediately after HAT to the ferryl state. The ability of the 2OG-dependent oxygenases to activate C-H bonds arises from the combined energy of the reduction potential (ERd) of the F4 state and the pKa of the resulting F3 state.1 The Raman evidence suggests the transient F3 intermediate is deprotonated, a particularly surprising finding that suggests that the pKa of this intermediate is low. Synthetic models have suggested that this pKa should be greater than 20,10 making it difficult to rationalize the formation of an alkoxide species in the AF mechanism. The free energy for HAT that leads to the formation of an alkoxide species via the Fe(III)-O– intermediate could be gained from either a highly positive ERd of the F4 state or a dynamic modulation of pKa of the proton donor/acceptor pair associated with the



subsequent proton transfer. The lack of experimental data in support of either one of these possibilities can be addressed by characterization of the redox, vibrational, and pH-dependent properties of the redox transitions between the in situ Fe(III) and Fe(IV) models of the transient F3 and F4 intermediates of TauD (Fig. 2). To better understand the F3 state, we have focused on characterizing the redox and pHdependent properties of the off-pathway Fe(III)-OH states for Fe-TauD, 2OG-Fe-TauD, and taurine-2OG-Fe-TauD. Unexpectedly, our initial investigation of the Fe(II)/(III) redox couples for these species revealed reversible, redox-linked conformational changes that modulate their redox potentials by at least 300 mV.11 Based on the observed vibrational changes and the magnitudes of the redox hysteresis, we proposed an isomerization that involves more than one primary metal ligand and the reorganization of the protein backbone. In this study, we use sitedirected mutagenesis of the primary Fe ligating residues, H99, D101, and H255 (Fig. 1), to test this hypothesis and to characterize the contributions of individual ligands to the overall redox response of the active site. Following time-dependent shifts in redox equilibria with chromogenic mediators, we demonstrate that isomerization is a reversible, multi-step process and show that the total magnitude of the electrochemical hysteresis is substantially larger than suggested by the initial spectro-electrochemical study. In addition, we demonstrate that redox hysteresis is not unique to TauD, but also is observed in the 2OG-dependent ethylene-forming enzyme (EFE)12 and may be a more general feature of this enzyme family. Experimental procedures Protein purification and spectroelectrochemical measurements Mutagenesis of the gene encoding TauD was performed as previously described.13,14 EFE apoprotein was purified by established procedures.12 Isolation of the TauD variant apoproteins, preparation of Fe(II) and Fe(III) holoenzymes of EFE and the versions of TauD, and spectroelectrochemical measurements were performed as previously described for wild-type (WT) TauD.11 Experimental Fourier transform infrared (FTIR) normal pulsed spectrovoltammetry (NPSV) data (Fig. S1) were deconvoluted into spectra and population profiles using the G3F package for Igor Pro.15 Chemical redox titrations


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Chemical redox titrations were performed under anaerobic conditions at room temperature. UV-Vis spectra were acquired using a Hewlett Packard 8453 spectrophotometer. A long-pass filter with a cutoff of 400 nm was used to suppress photochemical reduction of mediators. All samples were prepared in 25 mM Tris, pH 8, buffer. The reactions were initiated by addition of a 70% equivalent of anaerobic TauD or EFE to an anaerobic solution of reducing or oxidizing mediator in a stirred, anaerobic optical cuvette with 1 cm pathlength. Spectra were acquired for 1 h with progressively increasing time points of the sampling. Results Shifts in the E½ of TauD associated with redox-linked conformational changes can be probed by observable time-dependent changes during the equilibration of the enzyme with other redox active analytes, provided that the initial bimolecular reaction rate is faster than the ensuing protein reorganization. Whereas the visible optical absorption of TauD is too weak to detect these changes,16 the progress of the redox reactions can be followed by monitoring the redoxdependent optical changes in the spectra of mediators. Typical time-dependent absorption changes upon reduction of ferricyanide (FCN) by 2OG-Fe(II)-TauD (Fig. S2, left) show a multiphasic response. The initial fast kinetic phase (Table 1) arises from the redox equilibration controlled by the slower of two steps - the mediator binding and the electron transfer in the protein-mediator complex. Following that phase are multiple slower kinetic phases, which cannot be limited by the bimolecular rate and, therefore, are attributed to the changes in the properties of the protein. This behavior contrasts with a simple, monophasic oxidation of ferrous Mb by FCN,11 or the reduction of ferric myoglobin (Mb) by thionine acetate (TA, Fig. S3, left), as expected for a sample with reversible electrochemical behavior. Reactions with Mb quickly reach a redox equilibrium that corresponds to a E½ of -155 mV, in good agreement with the reported value of -153 mV17 and show little change after the first 5 s. The lack of change in the extent of oxidation of TA by Mb confirms that the sample remains anaerobic over a 1 h measurement and that gradual changes observed in TauD are not due to a slow ingress of atmospheric O2. Similar multiphasic changes were observed upon the reaction of 2OG-Fe-TauD in the ferrous and ferric states with several oxidized and reduced mediators (Fig. 3 and Fig. S2, right), including methylene blue (MB), methylene green (MG), and N,N,Nʹ,Nʹ-tetramethyl-p-



phenylenediamine (TMPD), in addition to FCN and TA. It can be seen in Fig. 3 that mediators resulting in a large extent of reduction of 2OG-Fe(III)-TauD show a small extent of oxidation of 2OG-Fe(II)-TauD and vice versa. The extent of the reaction correlates with the redox potentials of mediators, especially for FCN, TA, and MB, which exhibit single accessible redox transitions. FCN (E½ = 215 mV, n = 1) showed a fast initial oxidation of TauD to approximately 50%, followed by a slow oxidation to near completion. We observed no evidence that 2OG-Fe(III)TauD can be reduced by ferrocyanide to any detectable extent. MB (E½ = -236 mV, n = 2) showed the opposite behavior; it slowly reduced 2OG-Fe(III)-TauD (Fig. 3B) and showed little oxidation of 2OG-Fe(II)-TauD (Fig. 3A). TA (E½ = -197 mV, n = 2) was effective at partial reduction and oxidation of 2OG-Fe-TauD to levels intermediate between those of FCN and MB. Although the two-electron redox transitions of TA and MB theoretically could slow the kinetics

Figure 3. Normalized population kinetic traces of the amount of 2OG-Fe(II)-TauD oxidized or 2OG-Fe(III)-TauD reduced after addition into solutions containing various mediators. A) 2OG-Fe(II)-TauD was titrated into solutions containing 1.4-fold excess electron equivalents of the listed mediators, all in their oxidized forms. B) 2OG-Fe(III)-TauD was titrated into solutions containing 1.4-fold excess electron equivalents of the listed mediators, all in their reduced forms. Time zero indicates the point at which TauD was added to the sample. The reported E½ values of the mediators (vs. Ag/AgCl) are shown in parentheses. Similar results were observed at other redox equivalent ratios (Fig. S4).


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by requiring simultaneous reduction of two TauD molecules, the kinetics of the observed changes were distinctly multiphasic after the reduction or oxidation commenced. MG and TMPD, both of which have two accessible n = 1 redox transitions, showed similar trends. The E½ = +25 mV of TMPD was too high to be an effective reductant of 2OGFe(III)-TauD, whereas TMPD2+ caused nearly complete oxidation of 2OG-Fe(II)-TauD. The initial, large-amplitude oxidation was likely due to the TMPD/TMPD●+ redox couple (E½ = +25 mV), while the subsequent small-amplitude oxidation is due to the TMPD●+/ TMPD2+ redox couple (E½ = +425 mV). Similarly, the E½ = -111 mV redox transition of MG made it a more efficient oxidant of 2OG-Fe(II)-TauD than the structurally similar and more reducing MB and TA. Comparison of the reduction/oxidation efficiency of TA and MG also suggests that the n = 2 redox couple of TA was not a determining factor in its reactions with TauD. The smaller extent of reduction of 2OG-Fe(III)-TauD by MG than by TA, however, suggests that the E½ = -243 mV redox couple of MG is ineffective in reducing TauD. Finally, the exhaustive and monotonic reduction of 2OG-Fe(III)-TauD by methyl viologen (MV) (Fig. 3B) confirms that the entire population of TauD is redox active. This demonstration, along with the facile oxidation of TauD by FCN and TMPD allows one to attribute the transient kinetics observed with the structurally similar TA, MG, and MB to thermodynamic changes, rather than kinetic limitations. Table 1. Apparent initial rate constants of the oxidation (kOx) or reduction (kRd) of 2OG-Fe-TauD by various mediators.

Mediator

kOx (µM-1s-1)

kRd (µM-1s-1)

FCN

0.69

‒

TMPD

0.43

0.11

MG

0.11

0.35

TA

0.28

0.013

MB

‒

0.14

MV

‒

0.91

Since the bimolecular reactions of TauD with mediators (Table 1) are much faster than the ensuing slow changes in TauD and because the E½ values of the mediators are known (Fig. 3), the time-dependent E½ of TauD can be calculated using the Nernst equation. This analysis can be done following a single chromogenic component of the mixture and known initial



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concentrations of the mediator,  M 0 , and TauD,  TauD 0 . For the reduction of FCN by Fe(II)TauD, for example:  M  Ox t E½,TauD  t   ln   

TauD    M    M    RT  E  nF  M    M    Rd 0

Ox 0

Ox 0 2

Ox t

½, M

(1)

Ox t

where E½,M is the standard redox potential of FCN, and E½,TauD(t) is the time-dependent redox potential of TauD (see SI for derivation in general form). In this example, the initial (  M 0 ) and transient (  M t ) concentrations of FCN can be determined optically. The results of this analysis for the reduction of FCN by the WT protein and several variants of 2OG-Fe(II)-TauD are shown in Fig. 4 (top). The initial bimolecular reaction is fast (Fig. S2) and reaches equilibrium with approximately half of 2OG-Fe(II)-TauD oxidized to 2OG-Fe(III)-TauD prior to the first time point shown in Fig. 4. As 2OG-Fe(III)-TauD undergoes reorganization into a lower potential isoform, additional oxidation of 2OG-Fe(II)-TauD by FCN re-establishes equilibrium of the high potential isoform. This process promotes further oxidation of Fe(II)-TauD by FCN in an attempt to re-establish equilibrium. Ultimately, the calculated E½ at time t is the measure of the total oxidation of TauD as the result of several redox and structural equilibria, similar to an effective potential of a redox buffer consisting of several mediators. The opposite shift of equilibria occurring upon oxidation of TA (n = 2) by 2OG-Fe(III)-TauD is shown in Fig. 4 (bottom). Estimates of the time-dependent redox potentials (Fig. 4) must consider unavoidable errors that arise from i) pipetting precision and ii) the temporal stability of optical measurements (SI). In the case of WT TauD, changes in E½ were substantially larger than the estimated errors (shaded areas in Fig. 4). In contrast, the oxidation of H99A protein by FCN was nearly complete immediately after the start of the reaction, allowing only for an estimate of the upper limit of its E½ at

Structural origin of the large redox-linked reorganization in the 2

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