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Flavin C4a-OOH and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single crystal spectroscopic methods and in the 1.86 Å resolution x-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to x-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and the C4a-OO(H) adducts, and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the x-ray beam. The aerobic crystals then form either a C4a-OH or a C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.
Although flavins and flavoproteins were discovered in the 1930s, their remarkable functional diversity continues to be characterized. It is now estimated that up to 4% of microbial or eukarytotic proteins are flavoproteins, and more than 1200 flavoprotein structures are currently available from the Protein Data Bank. Flavin-dependent proteins catalyze a wide range of biochemical reactions including aerobic and anaerobic metabolism, light emission, photosynthesis, DNA repair, plant phototropism, regulation of biological clocks, and the activation of oxygen for hydroxylation and oxidation reactions (1). This diversity derives from the flavin isoalloxazine ring system, which is ideally suited for oxidative or reductive reactions involving one- or two-electron transfer to and from other redox-active centers, as well as reactivity with molecular oxygen. Moreover, the isoalloxazine ring system can act as an electrophile or a nucleophile forming covalent adducts with either protein residues or reaction intermediates at the C4a, N5, C6, and C8M positions (see Figure 2 for nomenclature). The flavin is also influenced by the protein active site, which extends the reaction diversity and facilitates catalysis along a particular flavoenzyme reaction coordinate. Thus, the interactions between the flavin, active site residues, and substrate molecules yield almost limitless combinations, and consequently remarkable diversity in flavoprotein function.
The reaction of molecular oxygen with reduced flavoenzymes is fundamental to all aerobic organisms and can be orders of magnitude faster or slower than the analogous reactions of flavins in solution (2). The outcome of the reaction also varies greatly, and has been used to segregate flavoenzymes into different classes. For example, the NAD(P)H-dependent monooxygenases cleave the dioxygen O-O bond with incorporation of one oxygen atom into an organic product and the other oxygen atom is then released as water. In contrast, the ubiquitous flavin-dependent oxidases use O2 as a two-electron, two-proton acceptor to produce H2O2. A transient C4a-hydroperoxy-flavin species has been established by spectroscopic methods in p-hydroxybenzoate 3-hydroxylase (3), p-hydroxyphenylacetate 3-hydroxylase (4), luciferase (5), and microsomal flavin-containing monooxygenase (6). A similar species has also been detected in oxidases via rapid kinetic studies of pyranose 2-oxidase (7), on a mutant form of NADH oxidase (8), and pulse radiolysis experiments with glucose oxidase (9). Because a C4a-oxygen adduct is so rarely observed in oxidases, some authors recently proposed that flavin reoxidation in these enzymes proceeds preferentially by an outer-sphere electron transfer process, rather than through formation of a C4a-hydroperoxide intermediate (10). Regardless of the mechanism or enzyme family classification, to date there is no structurally defined flavin C4a-oxygen adduct in the Protein Data Bank.
Choline oxidase (E.C. 126.96.36.199; choline-oxygen 1-oxidoreductase) from Arthrobacter globiformis catalyzes the four-electron oxidation of choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an obligatory enzyme-bound intermediate (11). In each of the oxidative half-reactions, a molecule of O2 is converted into H2O2. The midpoint reduction potentials for the FAD in choline oxidase are 211±2 mV and -65±2 mV for the FAD - FADsq and FADsq - FADH- couples, respectively (12). These values are among the highest determined to date for any flavoprotein, and are thought to be influenced by several active site characteristics, including a covalent linkage between the C8M position and His99. We recently determined the crystal structure of choline oxidase at 1.86 Å resolution under cryogenic conditions (13). A novel, but unexplained feature of the structure is the flavin cofactor, which exhibits a distorted isoalloxazine ring system and suggests the presence of a novel C4a-adduct. In this report, we provide evidence strongly suggesting that a C4a-OH or C4a-OO(H) adduct forms in an x-ray dependent process under cryogenic conditions. The essential insights derive from single crystal microspectrophotometry concurrent with x-ray diffraction collected recently at the new single crystal spectroscopy facility located at the National Synchrotron Light Source (see supplemental information and Figure S1), which is now available on a full-time basis to the general user population. The spectroscopic data also correlates very well with density functional theory (DFT) calculations and the high resolution x-ray crystal structure of the enzyme.
Oxidized choline oxidase from Arthrobacter globiformis strain ATCC 8010 was expressed from pET/codA in Escherichia coli and purified to homogeneity as described previously (13-15). Crystals of choline oxidase were grown aerobically by hanging drop vapor diffusion from 1.2 - 1.8 M ammonium sulphate and 10% v/v dimethylsulfoxide (DMSO) in 0.1 M Bis-Tris propane, pH 8.5. Single crystals were transferred from the mother liquor into a cryoprotectant solution consisting of 3.4 M sodium malonate, pH 7.0 and allowed to soak for two minutes prior to flash freezing in liquid nitrogen (13). Several independent crystals were used for x-ray diffraction data sets and for single crystal microspectrophotometry. X-ray diffraction data were collected at either the SER-CAT facilities (22-ID and 22-BM) at the Advanced Photon Source at Argonne National Laboratory, or at beamlines X12-B, X25, X26-C or X29 of the National Synchrotron Light Source at Brookhaven National Laboratory. The crystal structure was determined by molecular replacement as previously described (13) using the coordinates of the crystal structure of glucose oxidase (1CF3, (16)) as a search model. Choline oxidase crystallizes with one homodimer in the asymmetric unit in space group P43212 with a = b = 84.4 Å, and c = 343.5 Å. The high resolution data set extends to a resolution of 1.86 Å. Crystals of aerobic choline oxidase were also grown from 1.2 - 1.8 M ammonium sulfate and 10% v/v 1,4-dioxane in 0.1M Bis-Tris propane, pH 8.5. These conditions yield crystals with the space group P21 with typical unit cell dimensions of a = 69.3 Å, b = 346.2 Å, c = 105.9 Å, β = 94.3° and four homodimer enzymes in the asymmetric unit.
Refinements and model adjustments for the active site FAD were carried out as previously described (13). All library files with restraints were prepared using the PRODRG server (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/). Initially, the FAD was refined using restraints to confer planarity on the entire isoalloxazine ring. Electron density maps at this point clearly revealed significant bending of the pyrimidine ring and the mFo-DFc maps contoured at 3σ returned negative features on the flat pyrimidine ring and a lobe of positive difference density protruded from the C4a position and the pyrimidine ring. The isoalloxazine was then manually adjusted to properly fit the pyrimidine ring into the electron density and the restraints were adjusted to remove planar restraints from the pyrimidine ring and sp3 hybridization was conferred upon the C4a atom. Planar restraints were applied to the dimethylbenzene ring and the atoms of the piperazine ring. Electron density maps from the refinement indicated a good fit of the pyrimidine ring at this point, but the mFo - DFc maps revealed > 4σ positive difference features near the C4a atom. At this point, two series of refinements were performed. In one round, the sp3 C4a atom was bonded to a single oxygen atom (FAD-C4a-O-) and the model was refined. An FAD-C4a-O2- model was also refined. The resulting maps indicated that the O2 moiety also fits the 2mFo - DFc maps quite well; however, the occupancy of the distal oxygen atom refined best with an occupancy value of 0.5. To check for model bias, 2mFo-DFc and mFo-DFc simulated annealing omit maps were prepared in CNS. The FAD, DMSO, residues His99, and His466 as well as all atoms within a 3.5 Å radius were removed from the model. The simulated annealing and map calculation was carried out with a starting temperature of 1000 K with data from 50.0-1.86 Å resolution.
Two additional models were also refined with REFMAC5. The first included a water molecule centered in the difference peak and unrestrained with respect to the FAD C4a atom. Upon convergence of the refinement of this model, the water molecule was only 1.6 Å from the C4a atom (Table S1) and there was continuous electron density between the two atoms. However, since there is no H2O-C4a bond, this model does not satisfy the observed sp3 hybridization of the flavin C4a atom. The second alternative model consisted of a covalent FAD C4a-OH moiety, which refined to a 1.45 Å bond distance.
We built a Single Crystal μ-Spectroscopy Facility (SCμSF) at beamline X26-C at the National Synchrotron Light Source at Brookhaven National Laboratory for use by the general user population (see Figure S1). The microspectrophotometer components were from a 4DX-ray Systems AB (Sweden). We adapted it so that the microscopic optical axis and focal points were aligned with the crystal rotation axes of the diffractometer and x-ray beam at X26-C. The microscope objectives used parabolic mirrors to achieve 15x magnification and to minimize spherical or chromatic aberration in the wavelength range from approximately 150 - 10,000 nm. The objectives provide a 24 mm working distance through a 0.4 numerical aperture, which allows for cryocooling and access for other microspectroscopic components. When coupled with a 50-micron quartz optical fiber, the incident spot size is approximately 25 μm in diameter. The transmitted light is collected from a spot approximately 75 μm in diameter. The incident light (350 - 850 nm) was from a 75W Xe research arc lamp (Newport Corp.). An Ocean Optics USB 4000 spectrophotometer (Dunedin, Florida) containing a 3648-element Toshiba linear CCD detector was used to collect the optical absorption spectra. The data were processed initially with the SpectraSuit software on either a Windows XP or LINUX operating system. The spectrophotometer was calibrated and microspectroscopy aligned with a Hg-Ar calibration laser. Typically, optical absorption spectra were collected by averaging ten spectra, each of which was collected with an approximately 70 ms integration time and a 10-pixel “box car” of the CCD detector array. Crystals were held at 100 K during x-ray diffraction and optical absorption spectroscopic data collection.
All calculations were performed using the Gaussian 03 program (17). The geometries of all the structures were optimized without any symmetry constraints using Density Functional Theory (DFT) based B3LYP method with the 6-31G(d) basis set in gas phase (18-20). The final energies were calculated using a large 6-31+G(d,p) basis set including diffuse and polarization functions. Since it was computationally unfeasible to calculate unscaled zero-point energy and thermal corrections on the large models used in this study, they were not included. The dielectric effects (for ε = 4.3) from the surrounding protein were incorporated using the self-consistent reaction field method (21) at the B3LYP/(6-31G(d)) level. In order to retain the steric effect of the surrounding protein, one hydrogen atom each in the backbones of His351 and His466 residues were kept frozen from the X-ray structure. This kind of approach is known to preserve some of the steric effect of the protein surroundings (22). The remaining degrees of freedom of all the structures were optimized. The initial models used in the DFT calculations were extracted from the crystal structure. Rather large models were used in the calculations (ca. 180 atoms) and included the FAD and active site residues His99, Asn100, Ser101, DMSO, Ile103, His351, His466, Asn510, and Pro511. The backbone atoms of residues His351 and His466 and the ribityl side chain of the FAD were omitted from the calculation.
To investigate the single crystal spectroscopic properties of choline oxidase as a function of x-ray exposure, we harvested yellow crystals of oxidized enzyme from aerobic mother liquor. After transferring crystals to a cryoprotectant (3.5 M Na-Malonate), they were individually mounted in nylon loops and flash-cooled by plunging them into liquid N2. We performed several types of experiments with enzyme crystallized in space groups P43212 or P21 from ammonium sulfate. The exposure to 1 Å (12.398 KeV) x-ray photons (~4 × 1010 photon/s through a 200 μm diameter collimated beam) at 100 K was performed on stationary crystals or with crystals rotated through 180° at beamline X26-C of the National Synchrotron Light Source. The results are very reproducible and typical spectra are illustrated in Figure 1. The absorption spectrum of choline oxidase crystals at 100 K prior to x-ray exposure reveals two maxima centered at 460 nm and 485 nm. We and others have observed that single crystal spectra at low temperature are anisotropic and depend critically on crystal orientation, especially when planar chromophores such as flavins are present within the crystal (23, 24). For example, AMO has collected single crystal spectra from several oxidized flavoenzymes (data not shown) including nitroalkane oxidase, xenobiotic reductase A, cholesterol oxidase, and thioredoxin reductase, which all yield two resolved peaks near 450 nm. In most of these flavoenzymes and in choline oxidase in particular, the two maxima at 460 nm and 485 nm move up and down along with baseline excursions, as well as change in their relative intensities to each other as a function of the rotation angle. Thus, the single crystal spectra of choline oxidase are better resolved than for the enzyme in solution at 300 K (for a typical example, see Figure S2), which yields a peak at 450 nm and a shoulder at about 480 nm. After x-ray diffraction data collection, the optical spectrum of choline oxidase crystals yields a single absorption peak at approximately 400 nm. The difference spectra (after - before) clearly shows an absorption band with λmax at 400 nm and features that extend from about 510 nm to longer wavelengths. The 400 nm feature represents the vast majority of the flavin species present in the region of the crystal exposed to x-rays and is remarkably similar to spectra obtained from transient flavin C4a-OO(H) or C4a-OH intermediates (3-8). For example, these types of C4a adducts typically have a maximum absorbance at ~380 nm; however, in low temperature single crystals of choline oxidase the absorbance spectrum for the C4a adduct is red-shifted approximately 20 nm. The longer wavelength features at 510 nm indicate that a small fraction of the enzyme contains a FAD semiquinone species as previously observed for choline oxidase in solution (25).
We next evaluated the time-dependent process of adduct formation in a single crystal at 100 K. Spectra were collected every ten seconds from a stationary crystal of choline oxidase during exposure to the monochromatic synchrotron x-ray beam at X26-C. As illustrated in Figure 1B-C, the difference feature at 400 nm increases in an exponential process with a t1/2 of approximately 40 seconds. Thus, the appearance of the spectral feature at 400 nm is nearly complete within the time required to collect only a small fraction of the unique x-ray diffraction data (equivalent to less than 10° of crystal rotation), even at the relatively modest x-ray intensity of beamline X26-C of the NSLS.1 This process is also approximately commensurate with the decrease of the 460 and 485 nm features attributed to oxidized FAD (t1/2 of approximately 100 seconds, Figure 1C). Optical spectra from enzyme in solution at room temperature show that the flavin semiquinone species (i.e. one electron reduced) has a spectrum that overlaps that of oxidized FAD (25). In contrast, the hydroquinone FAD species (i.e. reduced by 2 electrons), or the C4a-adduct observed here, each have very little absorbance in the 450 - 500 nm region. Consequently, the exponential decay seen at 485 nm appears to be slower than the observed rate of formation of the 400 nm feature assigned to the C4a adduct. All taken together, the spectroscopic analyses of single crystals at low temperature show that the decrease in the concentration of oxidized FAD in the region of the crystal exposed to x-rays is correlated to an increase of the concentration of the C4a-adduct.
The atomic structure and 1.86 Å resolution simulated-annealing omit electron density maps for the FAD are shown in Figure 2. The flavin isoalloxazine ring is not planar, as anticipated for oxidized choline oxidase, or bent along the N5-N10 axis as is often observed in reduced flavoproteins (26). The electron density for the dimethylbenzene and piperazine rings indicates that each ring is flat and coplanar with one another. However, the plane of the pyrimidine ring is at an approximately 120° angle to the plane of the other two rings (Table S1). The electron density for the pyrimidine ring indicates that it adopts a “half-boat” configuration in which the C4a atom is approximately 0.5 Å above the pyrimidine ring plane. Moreover, throughout the refinement process a greater than 4σ positive difference peak associated with the C4a atom persisted. This feature clearly indicates that the C4a atom is sp3 hybridized and, therefore, that a covalent flavin adduct is present in the crystal structure. The electron density for the difference feature is large enough to accommodate only one or possibly two atoms in a covalently linked FAD C4a-adduct.
To our knowledge, the materials and aerobic crystallization conditions do not include any reagents that can readily form a C4a adduct with oxidized FAD. Moreover, none of the reagents used to crystallize the enzyme alter the optical spectrum of oxidized choline oxidase indicating that the reagents do not perturb significantly the FAD electronic environment (see Figure S2). In contrast, reduced flavins in the semiquinone or hydroquinone states do react with O2 and can form C4a-oxygen adducts (2, 9). Therefore, we followed a conservative approach and refined a FAD C4a-OH atomic model, which converged well with REFMAC5 to yield a 1.45 Å C4a-O bond distance (Figure 2A and Table S1). Next we modeled an O2 molecule bound to the C4a atom of reduced FAD. After refinement with REFMAC5 the C4a-Op and Op-Od bond lengths are 1.4 Å (Figure 2B and Table S1) with a C4a-Op-Od bond angle of 116°. Although the estimated coordinate error of the model is approximately 0.1 Å, these parameters agree well with those determined by quantum mechanical calculations for the model FAD-C4a-OOH intermediate in p-hydroxybenzoate 3-hydroxylase and phenol hydroxylase (27, 28), as well as the DFT calculations discussed below. Therefore, the C4a-OH and C4a-OO(H) models for the FAD adduct in choline oxidase have reasonable geometry for sp3 hybridization and the appropriate bond lengths and angles for a C4a-adduct. However, the observed electron density for the distal oxygen atom (Od) is weaker than for the proximal atom oxygen (Op) and, consequently, the former atom also has a higher B-factor. This suggests that either the Od atom may be partially disordered, possibly due to precession about the C4a-Op bond, or that only one oxygen atom is present in the adduct.
The simulated-annealing omit maps provide the most unbiased view of the FAD adduct. During this procedure, the FAD, DMSO, and C4a-adduct were omitted from the model. The resulting mFo - DFc and 2mFo - DFc electron density maps (Figure 2A-B) calculated with reflections between 50 and 1.86 Å resolution were superimposed with refined models containing either the C4a-OH or the C4a-OO(H) adduct. This analysis prompts us to conclude that the FAD in choline oxidase is most likely a C4a-OH (hydroxy) flavin adduct. However, as discussed above, we can not unambiguously rule out the C4a-OO(H) (peroxy or hydroperoxy) adduct. The resolution of the structure, quality of the refined atomic models, and the fits to the observed electron density for choline oxidase are comparable to those of the recently reported dioxygen complexes of cytochrome P450cam (29), naphthalene dioxygenase (30), superoxide reductase (31), homoprotocatechuate 2,3-dioxygenase (32), and amine oxidase (33).
The x-ray crystal structure shows that the active sites are completely sequestered within each subunit of the dimeric enzyme such that there is no direct access of bulk solvent to the FAD isoalloxazine ring. Nevertheless, a DMSO molecule, an additive in the crystallization solution, is observed in the solvent excluded cavity within each active site (Figure S3). In addition to the covalent bond between the dimethylbenzene ring and His99, several deduced hydrogen bonds stabilize the isoalloxazine ring configuration and the C4a-OH (or C4a-OO(H)) adduct. For example, the FAD pyrimidine ring forms a network of hydrogen bonds with protein backbone atoms from Asn100, Cys102, Ile103, and Asn512. In addition there are side chain interactions between the pyrimidine ring and Asn100 and Ser101. Consequently, nearly every atom of the isoalloxazine ring in this unusual configuration with the potential to participate in hydrogen bonds does so with either a protein residue2 or the DMSO. Furthermore, the structure indicates that the atoms of either C4a adduct are stabilized by hydrogen bonds with the side chains of His351 and Asn510. Finally, the DMSO molecule is located adjacent to the C4a adduct and also hydrogen bonds with the side chain of Ser101 and the FAD N5 moiety.
To gain a better understanding of the electronic structure of the unique isoalloxazine ring configuration observed in the x-ray structure of choline oxidase, we performed a number of DFT calculations. Each structure was optimized using Gaussian 03 program (17) at the B3LYP/6-31G(d) level in the gas phase. The final energies were calculated using a large 6-31+G(d,p) basis set, which included dielectric effects (for ε = 4.3) from the surrounding protein using the self-consistent reaction field (IEFPCM) method at the B3LYP/6-31G(d) level. The largest calculations (ca. 180 atoms) included the FAD isoalloxazine ring and most of the first shell residues comprising the active site (see Figure S3; His99, Asn100, Ser101, Cys102, Ile103, His310, His351, His466, Asn510, Pro511, Asn512, and a DMSO molecule). Several flavin electronic states within the enzyme active site were computed including: a) one-electron reduced flavin semiquinone (FADsq), b) a reduced flavin C4a hydroperoxy complex (FAD-C4a-OOH) c) a flavin C4a-hydroxy complex (FAD-C4a-OH), and d) flavin C4a-peroxy complex (FAD-C4a-OO-). The metrics for the observed x-ray structure and the DFT optimized structures for each flavin species are summarized in Table S1.
The DFT calculations unequivocally indicate that formation of the FAD C4a adduct, and the first shell hydrogen bond interactions with the flavin moiety are both necessary to reproduce the observed x-ray structure (Figures 2, S3 and S4). Unfortunately, the accuracy of the DFT calculations and the uncertainty of the crystal structure do not support a clear discrimination between the C4a-OH or C4a-OO(H) structures. In contrast, gas-phase optimizations of proteinfree flavin adducts yield less distorted FAD-C4a-OOH and FAD-C4a-OH structures (Figure S4-D). By comparison of these structures, we estimate that the energies required to stabilize the x-ray structure of the enzyme are ~29 kcal/mol for the FAD-C4a-OH adduct and 22 kcal/mol for the FAD-C4a-OOH adduct. Either of these total energetic contributions that stabilize the distorted flavin C4a adduct is consistent with the crystal structure and the numerous active site hydrogen bonds deduced between the distorted FAD and active site residues. In this context, our computational analysis suggests that the hydrogen bonding interactions of the flavin with the side chains His99, Asn100, Ser101, Cys102, Ile103, Asn510, His466 and His351 (Figure S3) are essential for the stabilization of the distorted flavin C4a adduct. This is underscored by our additional computations (not shown) suggesting that removal of any of these residues in silico yields a more planar flavin C4a-adduct.
We also considered alternative scenarios including degradation of the isoalloxazine ring system similar to that reported by Iwata et al. (34), but in this case being initiated by reduction of the flavin in the x-ray beam. Indeed, due to the formation of C4a=O double bond some of the DFT optimized models resulted in ring-open products similar to the 10a-spirohydantoin characterized by Iwata. However, we ruled out this hypothesis based upon structural and spectroscopic evidence. First, our high resolution electron density maps are of sufficient quality to establish sp3 and sp2 hybridization at the flavin C4a and C10a atoms, respectively. In contrast, the best characterized 10a-spirohydantoin degradation product has sp3 and sp2 hybridization at atoms analogous to flavin C10a and C4a, respectively. Our single crystal spectroscopic analysis also supports the C4a adduct. Indeed, while the optical spectrum for the choline oxidase adduct has a λmax near 400 nm, the optical spectrum for the degradation products referred to by Iwata have λmax near 310 nm.
Solvated electrons are generated in biological samples by synchrotron x-ray irradiation on the time scale of electronic transitions (see for example (35) and references therein). Rapid electron transfer over significant distances can be facilitated by aromatic side chains, protein backbone atoms, and protein secondary structure (36). Consequently, it is very likely that the FAD in choline oxidase is reduced in the x-ray beam. We propose that the high reduction potential for the enzyme bound FAD provides a strong thermodynamic driving force to capture the solvated electrons (Figure 3, path a). This conclusion is consistent with the negative features observed in the difference spectra at 450 and 480 nm, as well as the 510 nm features ascribed to the FAD semiquinone as a minor, but persistent species. Our DFT calculations indicate that the unpaired electron in an anionic FAD semiquinone radical, that is stabilized in choline oxidase, is delocalized between the C4a (0.19e), C9 (0.18e), C7 (0.08e), C8 (-0.13e) and C8M (0.58e) atoms of the isoalloxazine ring system. Thus, the spectroscopic results show that the exposure of the crystal to the x-ray beam at low temperature is commensurate with a decrease in concentration of oxidized FAD in the sample.
Hydroxyl and superoxide radicals, as well as other reaction products, are also generated in biological samples by x-ray exposure, but on a time scale of microseconds to milliseconds (35). However, the migration of hydroxyl and superoxide radicals, dioxygen, and protons within proteins at cryogenic temperatures is less well understood than electron transfer processes. Nevertheless, this raises the possibility that the C4a-adduct may form by one of several scenarios, which differ principally in the origin of the oxygen atom in the C4a-adduct (Figure 3). For example, our DFT calculations show that a hydroxyl radical does react with the C4a position of a FAD semiquinone yielding a flavin C4a-OH species, but only if the two radicals are near each other (ca. 2.0 - 2.2 ), and only if the flavin N5 is unprotonated (Figure 3, path b). Similarly, our calculations indicate that a reaction between superoxide radical and the FAD semiquinone yielding a flavin C4a-OO(H) species is also possible (Figure 3, path c). Alternatively, the C4a-OO(H) adduct may derive from a reaction between a two-electron reduced flavin (FADH-) and a ground-state O2 (Figure 3, path d). Previous experimental and theoretical studies have demonstrated that a flavin radical can react with dimethylsulfide to yield a flavin C4a-OH adduct and DMSO (37, 38). Our crystallization conditions include 10% v/v DMSO, which likely has dimethylsulfide as a minor contaminant component. Consequently, a trace amount of dimethylsulfide could promote conversion of the semiquinone to yield the C4a-OH adduct, but in this case the resulting oxygen atom in the observed adduct originates from O2 rather than solvent. Each of the scenarios presented here requires that for a C4a-adduct to form, the appropriate reactants (with the exception of solvated electrons) must be either present or generated in the vicinity of the flavin isoalloxazine ring. Indeed, diffusion of bulky species over long-distances or through the protein and/or crystal matrix to the interior FAD C4a position is very unlikely at 100 K. In contrast, the proposed short-range migration of radical species or ground-state O2 within the choline oxidase active site is analogous to the well documented results of carbon monoxide photodissociation of myoglobin-CO complexes (39, 40).
Irrespective of the mechanism of formation of the flavin C4a-OH or C4a-OO(H) adduct, it seems likely that once formed neither breaks down to release water or H2O2 because the cryogenic conditions do not establish the correct proton inventory on the surrounding residues and the flavin to facilitate product dissociation.3 In contrast, during catalytic turnover with choline (11), the enzyme coordinates the extraction of two protons and two electrons from the substrate with their subsequent delivery to O2 to yield H2O2 (see Figure S5). Indeed, during the reductive half reaction, the net transfer and storage of two proton equivalents from the substrate to the active site base and the flavin N5 atom is commensurate with the two-electron reduction of the flavin. In the subsequent oxidative half reaction, the two electrons are transferred from the reduced flavin to O2 along with the delivery of the two stored protons. Therefore, an incorrect proton inventory on the active site residues may stall the oxidative half-reaction. Thus, the x-ray exposure at low temperature creates an improper proton inventory scenario that consequently yields the structure of choline oxidase containing the C4a-adduct. To date, we have not obtained evidence for a similar C4a adduct species in choline oxidase in experiments conducted in solution. However, very recently Sucharitakul et al reported that pyranose 2-oxidase from Trametes multicolor does form a transient C4a-hydroperoxyflavin intermediate in its oxidative half-reaction. Moreover, that enzyme exhibits spectroscopic characteristics for the C4a-adduct in solution that are very similar to the adduct we observe in single crystals of choline oxidase. In addition, the pyranose 2-oxidase from Trametes multicolor (41-44) is structurally related to choline oxidase and other members of the Glucose-Methanol-Choline (GMC) oxidoreductase enzyme superfamily that catalyze the oxidation of alcohols.
Flavoenzyme mechanistic schemes often invoke FAD C4a-OO(H) or C4a-O(H) intermediates, but they have heretofore eluded structural characterization and have only rarely been detected by transient kinetic and spectroscopic techniques (1, 2, 9). However, the structural environment in the active site of choline oxidase appears to be ideally suited to stabilize a C4a adduct involving an oxygen species. This study is therefore the first direct observation by x-ray crystallography of a hydroxy-flavin or peroxo-flavin intermediate in any flavoenzyme, despite decades of effort by many researchers in the field, and numerous mechanistic proposals that invoke such a species. Our multidisciplinary experimental approach and correlation with complementary theoretical analysis thus provides direct evidence for an important oxygen intermediate in the reaction cycle of flavin-dependent enzymes.
Data for this study were measured, in part, at beamlines X12B, X25, X26C, and X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. Use of the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy Office of Basic Energy Sciences, under Contract DE-AC02-98CH10886. Some of the x-ray diffraction data was also collected at the Southeast Regional Collaborative Access Team (SER-CAT) beamlines 22-ID and 22-BM at the Advanced Photon Source (APS), Argonne National Laboratory (SER-CAT supporting institutions may be found at: www.sercat.org/members.html). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. We thank the SER-CAT staff at APS for assistance during data collection and Dr. Zhongmin Jin for assistance with the mail-in crystallography program. We are also grateful to the PXRR scientific staff at BNL for many helpful discussions, to Mr. Matt Cowan for assistance with the analysis and plotting of the time-dependent spectroscopic data of choline oxidase crystals, and to Ms. Mary Carlucci-Dayton for engineering and design assistance to mount the microspectrophotometer to beamline X26-C.
This work was supported in part by a grant from the American Chemical Society Petroleum Research Fund (40310-G4), an American Heart Association Grant in Aid (0555286B), by the Offices of Biological and Environmental Research of the US Department of Energy, and the NIH (2 P41 RR012408) to A.M.O.; by an NSF CAREER Award (MCB-0545712), a grant from the American Chemical Society Petroleum Research Fund (37351-G4), and a Georgia State University Research Initiation Grant to G.G., a Molecular Basis Disease Fellowship from Georgia State University to S.F., and a U.S. Department of Education GAANN Fellowship to G.T.L..
Data deposition footnote: The atomic coordinates and structure factors have been deposited to the Protein Data Bank with the file names 2jbv.
1We believe that the spectroscopic alterations we observe as a function of x-ray exposure are not simply related to the intensity of the synchrotron x-ray beam, but also to the total dose absorbed by the crystal during data collection and the active site environment. Therefore, the chemistry and x-ray dose dependence of adduct formation is rather complex and is currently under further investigation.
2In contrast, preliminary x-ray diffraction and structural analysis of an active site mutant of CHO where Ser101 was replaced with alanine does not yield an adduct with conditions that do form the adduct in the wild-type CHO.
3To date, we have not been able to determine cryo-conditions that allow us to thaw crystals after x-ray exposure and then permit us to use them for additional low temperature spectroscopic and diffraction studies.
SUPPORTING INFORMATION AVAILABLE Selected bond distances and angles in the x-ray structure and DFT optimized models (Table S1). The single crystal optical absorption spectroscopy facility installed at beamline X26-C at the NSLS (Figure S1). Optical absorption spectrum of choline oxidase in solution, at room temperature, and with all the crystallization reagents added (Figure S2). The active site environment of choline oxidase illustrating the potential hydrogen bonding interactions that stabilize the C4a-adduct (Figure S3). An overlay of several DFT optimized C4a-adducts and the crystal structure of choline oxidase (Figure S4). The proposed reaction mechanism for choline oxidase (Figure S5). The coordinates for several DFT optimized FAD C4a-adducts in X, Y, Z and PDB formats. This material is available free of charge via the Internet at http://pubs.acs.org.