Báo cáo khoa học: Deflavination and reconstitution of flavoproteins Tackling fold and function pot

REVIEW ARTICLE Deflavination and reconstitution of flavoproteins Tackling fold and function Marco H. Hefti*, Jacques Vervoort and Willem J. H. van Berkel Laboratory of Biochemistry, Wageningen University, the Netherlands Flavoproteins are ubiquitous redox proteins that are involved in many biological processes. In the majority of flavoproteins, the flavin cofactor is tightly but noncovalently bound. Reversible dissociation of flavoproteins into apo- protein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of the natural cofactor with artificial flavins has proved to be especially useful for the determination of the solvent acces- sibility, polarity, reaction stereochemistry and dynamic behaviour of flavoprotein active sites. In this review we summarize the advances made in the field of flavoprotein deflavination and reconstitution. Several sophisticated chromatographic procedures to either deflavinate or reconstitute the flavoprotein on a large scale are discussed. In a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain. Studies from riboflavin- deficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process. These genetic approaches have also provided insight into the mechanism of covalent flavinylation and the rationale for this atypical protein modification. Keywords: apoprotein; deflavination; FAD; flavin; flavo- enzyme;flavoprotein;FMN;(metal)affinitychromato- graphy; reconstitution. Introduction Flavoproteins are ubiquitous proteins that use flavins as prosthetic groups. The common flavin cofactors are FMN and FAD, which are synthesized in vivo from riboflavin (vitamin B1) by the action of riboflavin kinase [1,2] and FAD synthetase [3]. The redox active isoalloxazine moiety of the flavin cofactor may undergo one- or two-electron transitions [4]. This property and the ability to catalyse a wide range of biochemical reactions [5,6] make flavoproteins to be at the crossroads of cellular redoxchemistry. During the past 60 years, an impressive amount of flavoproteins has been characterized and many details about their cata- lytic and structural features have been determined. In this period, Vincent Massey was the leading character in flavin research [7]. In most flavoproteins, the flavin is tightly but noncova- lently bound. However, in a subset of flavoproteins, the flavin is covalently attached to the polypeptide chain [8]. Nature facilitated the binding of flavins to proteins by the evolution of different flavin binding folds [9–12]. Within these folds, specific parts of the flavin molecule are recognized by different structural motifs, like for instance the TIM barrel and the Rossmann fold. Many proteins that do bind FMN do not interact with FAD, and vice versa, and this is reflected in their fingerprint sequences [13–20]. Some flavoproteins contain both a FMN- and a FAD- binding domain. Well-studied examples of these diflavin enzymes include NADPH-cytochrome P450 reductase [21], nitric oxide synthase [22,23], flavocytochrome P450 BM3 [24], methionine synthase reductase [25,26], sulfite reductase [27–31], glutamate synthase [32,33] and dihydropyrimidine dehydrogenase [34]. In this paper, we present an overview of the field of flavoprotein deflavination and reconstitution. This topic is of central interest to flavin enzymology as it provides valuable insights in flavoprotein folding, function and mech- anism. Earlier reviews in this field have concentrated on the methods of apoflavoprotein preparation [35–37], the use of artificial flavins [38], and the functional role and mechanism of covalent flavinylation [8,39]. Here, new methods of flavoprotein reconstitution are described and combined with insights obtained from the structural and functional analysis of mutant enzymes. In the first part of this review, the thermodynamics of flavin binding and the value of flavin analogs are briefly discussed. Then, the old vs. new approaches of reversible flavin removal are evaluated. Finally, attention is given to the reconstitution of proteins containing covalently bound flavins. A comprehensive overview of the kinetics and thermodynamics of flavo- protein reconstitution is beyond the scope of this article and only selected cases are discussed. Correspondence to W. J. H. van Berkel, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, the Netherlands. Fax: + 31 317 484801, E-mail: willem.vanberkel@wur.nl Abbreviations: DAO, D -amino-acid oxidase; HAP, hydroxyapatite chromatography; VAO, vanillyl-alcohol oxidase; PHBH, p-hydroxy- benzoate hydroxylase; Nbs2, 5,5¢-dithio-bis(2-nitrobenzoate); IMAC, immobilized metal-affinity chromatography. *Present address: Key Drug Prototyping BV, Wassenaarseweg 72, 2333 AL Leiden, the Netherlands. Dedication: dedicated to Vincent Massey (1926–2002). (Received 17 June 2003, accepted 21 August 2003) Eur. J. Biochem. 270, 4227–4242 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03802.x Thermodynamics of flavin binding The binding interaction between apoproteins and flavin prosthetic groups has been studied extensively. The strong and specific binding of FMN or FAD to apoflavoproteins is driven by the enthalpic contribution to the free energy change of binding [40–42]. The thermodynamics of flavin binding to flavodoxin have been studied in detail [42–46]. Carlson and Langerman [44] showed that the enthalpy, entropy and free energy associated with the binding of FAD, 8-carboxyriboflavin, or the natural cofactor FMN to Azotobacter vinelandii apoflavodoxin were all negative. However, striking differences in the enthalpy for binding FMN, FAD and 8-carboxyriboflavin ()28.3, )16.6, and )14.0 kcalÆmol )1 , respectively) were observed. It was concluded that the difference in binding enthalpy between FMN and 8-carboxyriboflavin results from the phosphate group in FMN, which binds in a predefined binding pocket in the apoflavodoxin at the N-terminus of an a-helix. The unique hydrogen bonding network surrounding the FMN- phosphate group stabilizes the FMN-apoprotein complex [47]. This observation was recently corroborated by Lostao et al. [42], who dissected the binding energies of the Anabaena apoflavodoxin-FMN complex. It was shown that the contribution of the phosphate to the binding energy is the greatest (7 kcalÆmol )1 ), that the contribution of the isoalloxazine is around 5–6 kcalÆmol )1 , and that the ribityl side chain contributes only 1 kcalÆmol )1 . For flavodoxin from Desulfovibrio vulgaris it was found that riboflavin only binds to the apoprotein in the presence of inorganic phosphate (Fig. 1). Moreover, co-operative effects were observed linked to the binding of inorganic phosphate and the 5¢-phosphate of FMN [48]. It was proposed that phosphate binding induces a conformational switch, creating a population of apoflavodoxin that is capable of binding the isoalloxazine ring [49]. The thermodynamics of FAD binding to D -amino-acid oxidase (DAO) has been studied by Matteo and Sturtevant [40]. The free energy of binding was shown to be largely independent of temperature. However, the enthalpy and the entropy of the binding interaction were strongly tempera- ture dependent. In contrast to the binding of FMN to apoflavodoxin, where the entropy strongly opposes bind- ing, the binding of FAD to DAO is enforced by a large positive entropic contribution. It was proposed that this is due to a decrease in the exposure of nonpolar groups to the solvent and a smaller negative entropic contribution resulting from a tightening of the protein structure with losses in (vibrational) heat capacity when the coenzyme is bound. In general it is clear that (preformed) hydrogen bonding networks are important in flavin-binding, resulting in large negative enthalpy factors and that the entropic contribution to flavin binding can be either negative in rigid apoproteins (flavodoxins) or positive in more flexible apoproteins [40]. The enthalpic and entropic contributions of flavin binding are very important, when dealing with apoflavo- protein preparation. First the enthalpic contribution should be decreased in order to release the flavin from the holo- protein and second the entropic contribution in the prepar- ation of the apoprotein should be decreased. The enthalpic contribution in flavin binding can be decreased by the addition of solutes that interfere with the specific inter- actions between flavin cofactor and apoprotein. These solutes can be unfolding agents (urea, guanidinium hydro- chloride) which break hydrogen bonds, or monovalent anions (chloride, bromide) which influence protein hydra- tion. Phosphate and pyrophosphate can be used addition- ally [50] as these compounds can bind specifically in the phosphate or pyrophosphate binding pocket of FMN and FAD containing flavoproteins, thereby diminishing the binding interaction of the flavin prosthetic group with the apoprotein [45,51]. Often a combined effect of urea and bromide provides the best result in the preparation of apoflavoproteins. In those cases where the apoprotein is relatively unstable, it is important to reduce the entropic contribution in the preparation of apoflavoproteins. Many flavoproteins are less stable when they loose their cofactor and care should be taken when removing the flavin prosthetic group. An attractive deflavination approach is to bind the protein to a chromatographic support. This diminishes entropic contributions by increasing the rigidity of the apoprotein. Flavin analogs In order to gain insight into how the protein environment influences the reactivity of the flavin, it is desirable to remove the native prosthetic group in a, for the protein, nondestructive way. The flavin prosthetic group can be replaced with an artificial [38,52–56] or isotopically enriched analog [57–69]. Replacement with a flavin analog should result in the (functionally active) reconstituted holoprotein. FMN and FAD analogs can be synthesized conveniently from riboflavin, either chemically [1] or enzymatically [3], and can be isotopically enriched [70]. Fig. 1. Crystal structure of Desulfovibrio vulgaris flavodoxin. The protein is depicted in green, the riboflavin moiety of FMN in yellow and the 5¢-phosphate moiety in lime green. 4228 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Artificial flavins have proved very useful in the determin- ation of the solvent-accessibility, polarity, reaction stereo- chemistry, and dynamic behaviour of flavoprotein active sites [38]. Artificial flavins can be used to study the relative distance between particular flavin atoms and the protein, especially when using a series of flavins which are modified at a specific position, thus varying the Vanderwaals radius of the substituent. The solvent accessibility of the dimethyl- benzene and pyrimidine part of the isoalloxazine ring system can be probed by the introduction of a reactive (photo- active) group [71–76]. Artificial flavins can also be used to covalently link the flavin to the protein [77], or to determine the nature of amino-acid residues in the flavin binding site [52,72,78,79]. The chemical modification of the flavin molecule can have drastic influences on its reactivity, spectral properties or redox potentials. This provides a powerful tool to study the enzyme reaction mechanism and/ or the flavin–apoprotein interaction [38,80–82]. Reconstitution experiments with 8-mercaptoflavins are useful to get information about the polarity of the flavoprotein active site. Binding of 8-mercaptoflavins to apoflavoproteins may result in strong absorption spectral changes, indicative for the stabilization of either the benzoquinoid or thiolate anion form [38]. Crystal structures of several flavoprotein oxidases have confirmed the original conclusion [38] that in these enzymes, the benzoquinoid resonance form of the 8-mercaptoflavin is stabilized by a positive charge, localized near the N1–C2 region of the isoalloxazine ring. The absolute stereochemistry of flavoproteins can be determined by replacing the natural cofactor with 5-deaza- 5-carba-8-demethyl-8-hydroxyflavin [3,83]. Incorporation of this artificial flavin allows the chemical analysis of the stereochemistry of hydride transfer which is, for kinetic reasons, not possible with the natural flavin. The absolute stereochemistry has been assigned in this way for a large number of flavoenzyme reactions, with the crystal structure of glutathione reductase serving as the standard [3,83]. Flavin analogs modified in the ribityl side chain may also provide insight into flavoprotein function and mechanism [54,84–87]. For medium-chain acyl-CoA dehydrogenase, it was shown that the replacement of natural FAD by 2¢-deoxy-FAD reduces the activity of the enzyme about a millionfold [84]. This strongly supported the view that the 2¢-hydroxyl group of the flavin ribityl chain is involved in the stabilization of the partial negative charge of the carbonyl oxygen of the acyl-CoA substrate in the transition state [84,88,89]. Isotopically enriched flavins are suitable to get a detailed view into the molecular and submolecular structure of the protein-bound flavin molecule. 13 Cand 15 NNMRchemical shifts can reveal both p electron density, conformational changes and dynamic behaviour of the flavin moiety, as well as the presence of specific hydrogens at the carbon and nitrogen atoms investigated. 13 Cand 15 N have a natural abundance of 1.1% and 0.4%, respectively. Therefore, the flavoprotein has to be reconstituted with 13 C- and 15 N- enriched flavins. This approach gives a detailed electronic view of the single atoms of the isoalloxazine ring system of the flavin prosthetic group in the various redox states and of reaction intermediates formed [49,60,70,90,91]. In the flavo- proteins studied by NMR, it was observed that, as a rule, the isoalloxazine ring carries a negative charge at the N(1) atom in the hydroquinone state. Moreover, in most flavoproteins the isoalloxazine ring is observed to be nearly planar in both the oxidized and the two-electron reduced state. A beautiful example of the value of NMR spectro- scopy in flavoprotein research was the identification of Intermediate II of the bacterial luciferase reaction [90]. In this study the presence of C(4a)-hydroperoxyflavin could be established unambiguously, which was a major step forward in understanding flavoprotein function. Preparation and reconstitution of apoflavoproteins There are many methods for the preparation of reconsti- tutable apoflavoproteins. Some already exist for over 60 years, other are more recently developed. They are all based upon either weakening of flavin binding or stabilizing the apoprotein formed. Established strategies for protein deflavination include: lowering the pH, increasing the salt concentration, changing the solvent and increasing the temperature. As the final goal is to obtain highly reconsti- tutable apoprotein in high yield, the approach should be chosen with care. Prolonged exposure to non-native condi- tions, for instance by dialysis, can lead to irreversible denaturation of the apoprotein. Therefore, the time needed for flavin removal should be relatively short. As each (apo)flavoprotein has its own characteristics, several strategies for the reversible removal of flavins from flavoproteins have emerged. Initial deflavination protocols were based upon precipitation, partial unfolding, or dialysis of the protein [35]. More recent techniques focus on the binding of the protein to a chromatographic support, facilitating the removal of the flavin, and reconstitution of the apoprotein [36,92]. When studying the properties of apo or reconstituted flavoprotein, one needs to consider the side-effects of residual flavin in the endproduct. If replacement with a flavin analog is desired, residual natural flavin might influence the catalytic properties of the reconstituted enzyme considerably. The presence of residual flavin can even be more problematic when investigating the physical and spectroscopic properties of the apoprotein. Below we describe several apoflavoprotein preparation procedures, starting with conventional methods. Then we turn to the growing field of immobilization-based deflavin- ation methods in which one uses specific characteristics of the holo flavoprotein to obtain the corresponding apopro- tein. As a guide, the methods of apoflavoprotein prepar- ation are listed in Table 1. Conventional methods In 1935, Theorell was the first who reported that flavopro- teins could be reversibly resolved into their constituents apoprotein and prosthetic group. To weaken the binding of the flavin, Old Yellow Enzyme was dialysed at pH 2.7, thus releasing the noncovalently bound FMN [93]. A few years later, it was reported that D -amino acid oxidase (DAO) [94] and the yeast enzyme of Haas [95] could release their flavin in the presence of high concentrations of ammonium sulfate, a kosmotropic salt. Based on these initial findings Ó FEBS 2003 Flavoprotein resolution and reconstitution (Eur. J. Biochem. 270) 4229 a more common procedure for the preparation of apo- flavoproteins was developed by combining a low pH with a high ammonium sulfate concentration [96]. The acid ammonium sulfate precipitation method appeared to be especially useful for ÔrecalcitrantÕ flavoenzymes such as lipoamide dehydrogenase from pig heart [97] and glucose oxidase from Aspergillus niger [96] although with both enzymes partial irreversible denaturation occurred, and variable amounts of reconstitutable apoprotein were obtained. Strittmatter showed that addition of a high concentration of potassium bromide to the acid ammonium sulfate solution weakens the electrostatic interactions between the flavoprotein and the flavin. This increased the apoprotein yield of cytochrome b 5 reductase [98] and oxynitrilase [99]. Further addition of charcoal, to adsorb the free flavin, resulted in an even more efficient removal of the prosthetic group [55,100,101]. Treatment with trichloroacetic acid is another precipita- tion method to resolve flavoproteins into apoflavoprotein and prosthetic group. This easy to perform method was developed for flavodoxin [102] and works also very well for the related FMN-binding domain of cytochrome P450 BM3 [103]. The precipitated apo forms of both these proteins can withstand the extreme acidic conditions applied and dissolve readily at neutral pH. Many flavoproteins irreversibly aggregate at low pH. Therefore, a procedure of apoprotein preparation was developed based on dialysis against halide anions at physio- logical pH. Flavoproteins which bind the flavin prosthetic group rather weakly can be deflavinated using a high concentration of bromide ions [104–110]. Chloride is less chaotropic and therefore less effective in removal of the flavin [111]. Stronger chaotropes such as cyanide, cyanate and thiocyanate have been used as well, but with these nucleo- philic agents significant conformational perturbations pre- venting holoprotein reconstitution may occur [109,112,113]. Addition of a phosphodiesterase or phosphatase to dilute solutions of holoflavoprotein shifts the equilibrium to the apo form, because free FAD or FMN is hydrolysed to FMN and riboflavin, respectively. These reactions are relatively fast, but not very useful for large scale apoprotein preparation [114]. Moreover, care must be taken to remove the cleavage enzyme. Ultrafiltration [115] and gel filtration [116] are more efficient than dialysis for large scale apoflavoprotein pre- paration. This is especially important when using extreme conditions. Apoglucose oxidase, for instance, can be prepared by acidification to pH 1.4–1.8, followed by gel filtration in the presence of 30% glycerol [55,62,117]. Based on far-UV circular dichroism data it was suggested that under these conditions, the apoprotein retains a compact fold with a high degree of native-like secondary structure [118]. Nevertheless, reconstitution of apoglucose oxidase with FAD is incomplete and about 50% of irreversibly aggregated apoprotein is generally obtained [62,117,119]. The reconstitution of apoglucose oxidase with FAD-analogs is of substantial importance for the construction of enzyme- electrodes which can be used as biosensor devices [120]. Table 1. Procedures of apoflavoprotein preparation. Conventional methods Chromatographic methods Acid ammonium sulfate precipitation Ion exchange chromatography Glucose oxidase [96] Egg white riboflavin binding protein [140,141] Lipoamide dehydrogenase [97] Egg yolk riboflavin binding protein [142] Trichloroacetic acid precipitation Hydrophobic interaction chromatography Flavodoxin [102] Lipoamide dehydrogenase [128,151,153] FMN domain cytochrome P450 BM3 [103] Glutathione reductase [150] Dialysis Mercuric reductase [150] With phosphodiesterase Butyryl-CoA dehydrogenase [150] p-hydroxybenzoate hydroxylase [114] DNA photolyase [152] With guanidinium hydrochloride L -Amino-acid oxidase [160] Lipoamide dehydrogenase [121] Flavocytochrome b2 [60] Cytochrome P450 BM3 [122] Hydroxyapatite chromatography With urea p-Cresol methylhydroxylase [165] Salicylate hydroxylase [123] Vanillyl-alcohol oxidase [166,167] Lactate oxidase [124] Dye affinity chromatography Cytochrome P450 reductase [21] Flavocytochrome b2 [172] With halide ions D -Amino-acid oxidase [173] Old yellow enzyme [93] p-Hydroxybenzoate hydroxylase [174] D -Amino-acid oxidase [104] Covalent chromatography Cytochrome b5 reductase [98] p-Hydroxybenzoate hydroxylase [114,239] Oxynitrilase [99] Immobilized metal affinity chromatography Xanthine oxidase [130] NifL PAS domain [69] Gel filtration Glucose oxidase [117] Carbon monooxide dehydrogenase [137] Hydroquinone hydroxylase [116] Ultrafiltration Cytochrome P450 reductase [115] 4230 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Another conventional procedure of apoprotein prepar- ation is to partially unfold the holoprotein with guani- dinium hydrochloride [121,122] or urea [21,123,124]. A disadvantage of this method is that one needs to find conditions in which the partially unfolded apoprotein is capable of refolding. Circular dichroism spectroscopy [46,118,125–127] and fluorescence spectroscopy [127–129] are useful here to probe the folding behaviour of the protein of interest. For many metalloflavoproteins, most of the conventional apoflavoprotein preparation procedures cause extensive denaturation. A likely explanation for this is that deflavin- ation and reconstitution of flavoproteins is difficult if the quaternary structure is more complex, i.e. when the protein contains more cofactors and/or subunits. The molybdo- iron-sulfur flavoprotein xanthine oxidase [130,131] can be deflavinated by dialysis at physiological pH in the presence of 2 M calcium chloride [132]. The FAD released is hydrolysed to FMN by the high concentration of salt present. Other methods are based upon this technique, and include calcium dicarbonate [133] or potassium iodide [134]. Carbon monoxide dehydrogenase from Oligotropha carb- oxidovorans [135,136] is another molybdo-iron-sulfur flavo- protein for which a specific procedure of flavin exchange was developed [137]. The flavoprotein subunits can be removed from this a2b2c2 enzyme by dissociation with sodium dodecyl sulfate. The recombinant flavoprotein component of carbon monoxide dehydrogenase produced in Escherichia coli is a fragile monomer that does not bind FAD. However, when the monomeric apoflavoprotein is complexed with the metalloprotein components, the result- ing heterohexameric form of the enzyme readily integrates FAD [137]. This shows that the reconstitution of the native enzyme involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Chromatographic procedures For several flavoproteins, the conventional methods of apoprotein preparation are satisfactory (Table 1). A good example is the trichloroacetic acid precipitation procedure developed for flavodoxins [102]. Multidimensional NMR studies have shown that the extreme conditions for apoflavodoxin preparation do not lead to any structural perturbation in the reconstituted holoprotein, compared to the native protein [138,139]. With many flavoproteins, and especially when large amounts of apoprotein are required (affinity-based) chromatographic procedures are the meth- ods of choice. One of the advantages of these methods is that on-column protein aggregation is unlikely to occur, as each molecule is at a relatively large distance from its neighbours. Particularly during partial unfolding, this helps stabilizing the apoform of the protein, before flavin reconstitution. Ion-exchange chromatography Many flavoproteins can be reversibly adsorbed to an ion- exchange support. However, for successful on-column flavin removal, conditions are needed where the protein still interacts with the ion-exchanger (low ionic strength) but not with the flavin (low pH). This concept was first worked out for the riboflavin-binding proteins from chicken egg white [140,141] and chicken egg yolk [142,143]. The holo forms of these carrier proteins can be separated in the apo forms and free riboflavin by cation-exchange chromatogra- phy at pH 3.7 [144]. At this pH, riboflavin is released from the column whereas the apoprotein remains tightly bound. The apoprotein can subsequently be released from the column by raising the pH and ionic strength of the elution buffer. Unlike most other flavoproteins, aporiboflavin-binding protein interacts strongly with a large number of flavin derivatives but not with FMN or FAD [141]. The structure of chicken egg white riboflavin-binding protein is rather unusual [145]. Besides from an N-terminal ligand-binding domain that is strongly conditioned by nine disulfide cross- links, it contains a flexible phosphorylated motif with nine phosphoserines, which is essential for vitamin uptake [146]. The isoalloxazine ring of the tightly bound riboflavin molecule is stacked in between two tryptophans, explaining the strong fluorescence quenching observed upon flavin binding. The interaction between aporiboflavin-binding protein and riboflavin in the oxidized and two-electron reduced state has been addressed by reconstitution of the protein with 13 C- and 15 N-enriched riboflavin derivatives [147]. These studies revealed that the pK a of the N1 atom of the flavin in the reduced state is unusually high (pK a ¼ 7.45). The effective binding of riboflavin to the apoform of riboflavin-binding protein has also received biotechnolo- gical attention. Aporiboflavin-binding protein was shown to scavenge riboflavin in model beer solutions, thereby inhibiting the light-induced formation of reactive oxygen species and sunstruck off-flavour [148,149]. Hydrophobic interaction chromatography To circumvent severe protein loss using conventional techniques, a generally applicable immobilization procedure was developed for the large scale preparation of apoflavo- proteins using hydrophobic interaction chromatography (HIC) [150]. This method makes use of the fact that many flavoproteins bind to phenyl agarose at neutral pH in the presence of 1 M ammonium sulfate. Entropy is the driving force in this process. After immobilization, the flavin can be removed by the addition of high concentrations of potas- sium bromide and/or lowering the pH of the elution buffer. The HIC method has been successfully applied for a number of flavoproteins [150,151], sometimes with slight modifications, to get optimal results [37,60,152]. The HIC method for preparing apoflavoproteins works very well for prokaryotic and eukaryotic disulfide reduc- tases, including lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, and is preferred over classical methods [150]. For apolipoamide dehydrogenase it was shown that the kinetics of holoenzyme reconstitution are dependent on the source of enzyme [151] and on the type of flavin [153]. Initial FAD binding to the monomeric apoprotein results in dichlorophenol-indophenol activity and quenching of tryptophan fluorescence. Then, dimeriza- tion occurs as reflected by the lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of Ó FEBS 2003 Flavoprotein resolution and reconstitution (Eur. J. Biochem. 270) 4231 the visible absorption spectrum [151]. For lipoamide dehydrogenase from A. vinelandii, the conformational sta- bility of the monomeric apoprotein was compared with that of the dimeric holoenzyme [128]. Unfolding of the apo- enzyme in guanidinium hydrochloride follows a simple two- state mechanism and is fully reversible. However, the midpoint of unfolding of the monomeric apoprotein (C m guanidinium hydrochloride ¼ 0.75 M ) is much lower than that of the dimeric holoenzyme (C m guanidinium hydro- chloride ¼ 2.4 M ). Guanidinium hydrochloride unfolding was also used to probe the conformational stability of A. vinelandii lipoamide dehydrogenase in the different redox states [128]. From this and additional mutagenesis studies it was inferred that overreduction by NADH promotes subunit dissociation and that the C-terminus of the protein plays an important role in dimer stabilization [154–156]. Sometimes the apoprotein interacts very strongly with the HIC column material and apoprotein elution or on-column reconstitution can be difficult. In such cases, the apoprotein may undergo irreversible conformational damage, e.g. by local unfolding or subunit dissociation. This was observed to some extent with the apoprotein of butyryl-CoA dehydrogenase from Megaspheara elsdenii [150], which can not be reconstituted when bound to the column. By using high concentrations of ethylene glycol as eluent, stable apoprotein showing negligible residual activity could be isolated with 50–80% yield. Spectral analysis of the apoprotein revealed that the coenzyme A persulfide ligand present in the native protein [157] is removed during apoprotein preparation. At pH 7.0 and 25 °C, the apopro- tein is a mixture of dimers and tetramers, and reassociates to a native-like tetrameric form in the presence of FAD. The reconstitution with FAD is relatively slow, and is stimulated in the presence of CoA ligands. Binding of CoA ligands stimulates tetramerization of the reconstituted holoenzyme and improves protein stability. This is in agreement with the crystal structure of butyryl-CoA dehydrogenase [158] which shows that the inhibitor acetoacetyl-CoA binds in an extended conformation near the dimer–dimer interface. Fluorescence/polarization experiments revealed that the reconstituted protein is somewhat less stable than the native holoprotein, and that FAD dissociates more easily [150]. Another protein that was successfully deflavinated by the HIC method is L -amino-acid oxidase from the venom of Crotalus adamanteus, the eastern diamondback rattlesnake [159]. L -amino-acid oxidase is a dimeric glycoprotein, containing one FAD per monomer, that catalyses the oxidative deamination of L -amino acids. The deflavination method for L -amino-acid oxidase [160] is similar to the original protocol developed for lipoamide dehydrogenase. The apo form of L -amino-acid oxidase remains bound to the HIC column, while the FAD cofactor is washed away in a buffer with 1.5 M ammonium sulfate. The apoprotein of L -amino-acid oxidase can be reconstituted with FAD on-column. However, the FAD-reconstituted L -amino-acid oxidase is inactive, having a perturbed conformation of the flavin binding site. In the presence of 50% glycerol the reconstituted L -amino-acid oxidase becomes nearly com- pletely active. It was suggested that repulsion of glycerol from hydrophobic surfaces of the protein and the simulta- neous interaction with protein polar regions initiates the restoration of the internal protein hydrophobic core. Thus, glycerol can have a restorative effect on the proposed partially unfolded equilibrium intermediates, and acts as a molecular chaperone [160]. This function of glycerol was first demonstrated for the His30Leu mutant of pig kidney DAO [161,162]. The replacement of His307 with leucine perturbs the active site conformation accompanied by weakening the protein–flavin interaction and decreasing the enzymatic activity. The negative effect of this mutation can be eliminated in the presence of glycerol, resulting in up to 50% activity recovery and more than 16-fold increase of flavin affinity. From this it was concluded that glycerol assists in the rearrangement of the protein towards the holoprotein conformation, as well as in reducing the solvent accessibility of the protein hydrophobic core [163]. Hydroxyapatite chromatography Hydroxyapatite chromatography (HAP) is often used as a final step in protein purification. For preparation and reconstitution of apoflavoproteins, HAP has the advantage that high salt concentrations, which can stimulate apo- protein formation, are not necessarily a limitation. The interaction between the protein and hydroxyapatite is primarily the result of non-specific electrostatic interactions between the positively charged protein amino groups and the negatively charged column material [164]. The deflavi- nation process is influenced by the charge distribution of the protein, as well as the kind of salt that is used as eluent. The HAP method of apoflavoprotein preparation was used for a recombinant form of the flavin-binding subunit of p-cresol methylhydroxylase from Pseudomonas putida [165] and for the His61Thr variant of vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum [166,167]. Binding the His61Thr variant to hydroxyapatite appeared to be a very gentle and efficient method of obtaining the VAO apopro- tein. Upon washing with 200 m M phosphate buffer, the His61Thr protein remains tightly bound to the column, whereas the FAD is easily removed. This removal of FAD is superior to other methods. For instance, when the His61Thr holoenzyme is gel filtrated in the absence or presence of high salt, almost no flavin is released. Another advantage of the HAP method is that the His61Thr VAO apoprotein can be eluted in concentrated form by washing the column with 600 m M potassium phosphate buffer. While native VAO forms primarily octameric assemblies of 507 kDa, the apoHis61Thr variant exists in solution as a dimer of 126 kDa. Binding of FAD or ADP to the dimeric apoenzyme induces octamerization. In contrast, incubation with riboflavin or FMN does not stimulate octamer assembly, suggesting that upon FAD binding, small conformational changes in the ADP-binding pocket of the dimeric VAO mutant are transmitted to the protein surface, thus promoting oligomerization [167]. Dye-affinity chromatography Polyaromatic dyes can bind to proteins that use a cofactor with a nucleotide moiety [168]. Cibacron Blue has a strong affinity for the Rossmann bab dinucleotide binding fold [125,169], and many flavoproteins possess such a fold, either for binding the ADP part of FAD and/or NAD(P) [170]. This feature can be used to separate apo and holo 4232 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003 flavoproteins [171]. Such a separation was performed with Cibacron Blue Sepharose for flavocytochrome b2 [172], and for DAO [173]. Red-A agarose, another polyaromatic dye-containing material, has been used for the deflavination of p-hydroxy- benzoate hydroxylase (PHBH) from Pseudomonas fluores- cens [174]. Under low ionic strength conditions, the red dye of the column binds to the enzyme, displacing the FAD. The column is then eluted with high-ionic strength buffer containing the artificial flavin 6-azido-FAD, which binds totheproteinanddisplacesthedye.The6-azido-FAD cofactor can be covalently linked to the protein by irradiation. Enzyme that has not been photolabeled is separated from the covalently photolabeled enzyme by applying the reaction mixture to a Red-A column again. At low ionic strength, the nonlabeled enzyme binds to the column material, whereas the photolabeled enzyme passes directly through the column [77,80]. Covalent chromatography When partial unfolding of the holoprotein is required to weaken the protein–flavin interaction, the above mentioned chromatographic methods for protein deflavination may not work properly because of the presence of high concentrations of unfolding agents. Therefore, we intro- duced the concept of covalent enzyme immobilization for improving the yield and quality of the apoprotein of PHBH from P. fluorescens [114]. PHBH from P. fluorescens is a homodimeric FAD- dependent monooxygenase that contains 5 sulfhydryl groups per monomer [175]. Cys116 is the only solvent exposed thiol group, accessible to N-ethylmaleimide and 5,5¢-dithio-bis(2- nitrobenzoate) (Nbs2) [176]. Using this property, it is possible to bind the enzyme covalently to a Nbs2–agarose column [114]. Oxidation [92] or mutation [177] of Cys116 does not influence catalysis but prohibits binding of the protein to the Nbs2 column. In Fig. 2, the PHBH dimer is shown together with the solvent accessibility of the Cys116 sulfur atom. After coupling to the column material, the FAD can be efficiently released from the protein with urea and KBr. The resulting apoprotein is eluted from the column after reaction with dithiothreitol. On-column reconstitution of the apoprotein with an artificial or isotopically labelled flavin analog is possible as well. The high resolution crystal structure of PHBH reconstituted with arabino-FAD [54] has clearly shown that the covalent disulfide-exchange procedure of apoprotein preparation is very mild and does not lead to any significant structural perturbation. The thiol affinity chromatography method requires the presence of a freely available thiol group at the protein surface, making the method not generally applicable. Although the introduction by mutagenesis of a surface accessible cysteine is in principle possible with every flavoprotein, this approach might sometimes be a long shot. Furthermore, the newly introduced cysteine should not interact intra or intermolecularly with other cysteines. For preparation of apoPHBH, the original Nbs2-agarose has recently been replaced by commercially available thiopropyl sepharose [81,178,179]. Immobilized metal-affinity chromatography Recombinant DNA technology has opened the possibility to add affinity tags to proteins, facilitating protein purifi- cation. In immobilized metal-affinity chromatography (IMAC), a polyhistidine tag at either the N-terminal or C-terminal end of the protein allows the strong binding of the protein to the column bound metal ion. This interaction is only disrupted by high concentrations of imidazole, acidic pH or chelating agents such as EDTA. A his-tagged flavoprotein bound to an IMAC column is in principle able to withstand rather harsh conditions that can be used to successfully remove the flavin prosthetic group. The IMAC method of apoflavoprotein preparation was developed for the flavin-containing PAS domain of NifL, a redox-sensing protein from A. vinelandii [180,181]. Deflavi- nation was achieved on a nickel-nitrilotriacetic acid column by exploiting the available N-terminal His-tag [69]. Protein- bound FAD was removed efficiently by washing the column Fig. 2. 3D-structure of PHBH. The FAD cofactor is shown in green. In the right panel, all amino-acid residues within a distance of 10 A ˚ to the Cys116 sulfur atom (yellow) are shown. The solvent accessibility of the sulfur atom is indicated with yellow dots, and Cys116 is drawn with Vanderwaals-radii. Ó FEBS 2003 Flavoprotein resolution and reconstitution (Eur. J. Biochem. 270) 4233 with KBr and urea. The apoprotein could be eluted from the column with imidazole, but slowly precipitated after column release. Therefore, on-column reconstitution was performed by circulating a solution of 2,4a- 13 C 2 -FAD or 2,4a- 13 C 2 -FMN (Fig. 3). The reconstituted PAS domain containing a new flavin cofactor was eluted from the column with imidazole. Reconstitution of the flavoprotein is highly efficient, as shown by NMR and UV-VIS absorption spectroscopy [69]. Due to the immobilization of the apoprotein during the process of de- and reflavination, no severe protein loss is observed. Although the NifL PAS domain normally binds FAD, the His-tag reconstitution method unambiguously showed that this protein also tightly interacts with FMN. 13 Cand 31 P NMR experiments confirmed that there is no significant difference in the active site between the FAD- or the FMN- reconstituted protein in either the reduced or oxidized state [182]. The use of IMAC for the preparation and reconstitution of His-tagged apoflavoproteins might become a general method as His-tag incorporation in recombinant proteins is easily achieved. Deflavination of covalent flavoproteins Most flavoproteins bind their cofactor in a noncovalent mode. However, in about 10% of the flavoproteins, the isoalloxazine ring of the flavin is covalently linked to either a histidine, tyrosine, or cysteine residue [8,39]. Members of the VAO family [18] have a remarkable tendency for covalent anchoring of the flavin cofactor, whereas flavoproteins with a Rossmann fold [10,183] prefer a noncovalent flavin binding mode. Succinate dehydrogenase [184–186], fuma- rate reductase [187,188], and monoamine oxidase [189–191] are the classical examples of Rossmann fold enzymes that contain a covalently bound flavin cofactor. Several strategies have been used to get insight into the mechanism of covalent flavinylation. One of these strategies, developed for human monoamine oxidase, makes use of a yeast strain auxotrophic for riboflavin and expression of the enzyme in this strain in the presence of different riboflavin analogs [192,193]. Another more generally applied strategy is based on the replacement of crucial amino-acid residues by site-directed mutagenesis [194–198]. In short, these investigations have supported early proposals from model system studies [199,200] that covalent flavinylation involves an autocatalytical iminoquinone-methide addition mechan- ism with flavin binding preceding covalent attachment [8,39]. In line with this mechanism it was found that the apoenzyme of monoamine oxidase B, expressed in COS cells devoid of riboflavin, is correctly inserted into the outer mitochondrial membrane [201]. For succinate dehydrogenase from yeast it was esta- blished that flavinylation takes place within the mitochond- rial matrix after import of the flavoprotein subunit and the cleavage of a leader peptide [186,202,203]. Moreover, flavinylation of this iron-sulfur flavoenzyme was enhanced in the presence of the chaperone protein hsp60 [186]. p-Cresol methylhydroxylase is an 8a-O-tyrosyl-FAD containing flavocytochrome involved in the anaerobic microbial degradation of alkylphenols. From individual expression of the heme- and flavin-binding subunits it was revealed that the apoflavoprotein component of p-cresol methylhydroxylase is capable of noncovalently binding FAD but that the interaction with the heme-containing subunit is required for the self-catalytic flavinylation reac- tion [204,205]. The rationale for covalent flavinylation is not always clear. It has been proposed that the covalent linkage is involved in (a) stabilization of the apoprotein structure (b) steric alignment of the cofactor in the active site to facilitate catalysis (c) modulation of the redox potential of the covalent flavin [39] and (d) suppression of unwanted side reactions [206]. For fumarate reductase from E.coliit was found that the replacement of His44 by either Arg, Cys, Ser or Tyr results in correctly assembled protein variants that are fully saturated with noncovalently bound FAD [207,208]. Based on the diminished activity with fumarate and the inability to oxidize succinate, it was suggested that the absence of the covalent linkage alters the redox potential of the flavin. More recently, this proposal received experi- mental support from studies on other flavoproteins [209,210]. For VAO from Penicillium simplicissimum it was shown that the covalent linkage between the C8a atom of the isoalloxazine ring of the flavin and the N3 atom of His422 increases the redox potential of the flavin, thereby facilitating substrate oxidation [209]. Furthermore, from crystallographic analysis of noncovalent VAO variants and the VAO apoprotein, it could be established that the flavin binds in this enzyme to a preorganized binding site and that His61 activates the neighbouring His422 for covalent binding of FAD [166,209] (see Fig. 4). A new type of flavin attachment was recently reported for the NqrB and NqrC subunits of the Na + -translocating NADH-quinone oxidoreductase from Vibrio alginolyticus [211,212]. From MALDI-TOF MS analysis of proteolytic digests, it was concluded that in both NqrB and NqrC subunits, the FMN cofactor is attached by its 5¢-phosphate moiety to a threonine side chain. In agreement with this, no covalent flavin was detected in the Thr225Leu mutant of NqrC from Vibrio cholerae [213]. From sequence compari- sons it was predicted that this novel type of phosphoester Fig. 3. Schematic representation of the preparation and reconstitution of His-tagged apoflavoproteins by immobilization on a nickel-nitrilotri- acetic acid column (from [69], with permission). 4234 M. H. Hefti et al.(Eur. J. Biochem. 270) Ó FEBS 2003 binding between FMN and the apoprotein is conserved in the NADH-quinone oxidoreductase sodium pumping sys- tems of a number of marine and pathogenic bacteria and that in some of these systems the target threonine is replaced by a serine residue [214]. Conclusions and future perspectives Since the pioneering work of Theorell [93], many methods have been developed for the (large scale) preparation and reconstitution of apoflavoproteins. Conventional precipita- tion methods are rapid but the yield and reconstitutability of apoprotein may vary dramatically. More recently developed chromatographic procedures have the advantage that the apoprotein is stabilized by immobilization, and that large amounts of apo- or reconstituted flavoproteins can be obtained. His-tagged flavoproteins can be purified, deflavinated and reconstituted on the same IMAC column. Therefore, the use of IMAC for flavoprotein deflavination and reconstitution should be further exploited. An interesting possibility to facilitate flavin release with such a column is to deflavinate the protein in its reduced state. In the reduced form, flavin binding usually is less tight than in the oxidized form [215,216]. The introduction of a solvent exposed cysteine residue is another engineering strategy that allows the covalent binding of a flavoprotein to a solid support. This method produces high quality apoflavoproteins with- out affinity tags in very good yield [114]. Site-directed mutagenesis can be used to change the strength of the flavin–apoprotein interaction. With such approach, it is important that the amino-acid replacements (or deletions) do not result in dramatic changes in protein stability and/or enzyme catalysis. For instance, a surface loop of 14 amino-acid residues was removed from Rhodotorula gracilis DAO by rational design [217]. This shifted the protein from the dimeric to the monomeric state. In the monomeric state, the FAD interacts more weakly with the protein. The DAO mutant apoprotein was then obtained by dialysis in the presence of a chaotropic agent [106]. Sometimes, site-directed mutagenesis is necessary to improve the flavin–apoprotein interaction. With L -aspartate oxidase, a FAD-dependent enzyme involved in the micro- bial biosynthesis of NAD + , the protein could only be crystallised in the apoform [218]. Attempts to crystallise the wild-type enzyme in the holo form were unsuccessful. However, the Arg386Leu mutant, in which an active site arginine is replaced, turned out to be amenable to crystal- lization and structure elucidation in the active FAD-bound state [219]. Understanding the specific interaction between flavopro- teins and their cofactors is also of medical relevance. Since the earlier finding that a reduced affinity of human glutathione reductase for FAD due to a mutation can lead to nonspherocytic haemolytic anaemia [220], several genetic defects affecting flavin binding have been described. Muta- tions causing impaired flavin binding have been reported for, e.g. NADPH-oxidase [221–225], NADH:cytochrome b 5 reductase [226–228], methylenetetrahydrofolate reductase [229–234], and dihydropyrimidine dehydrogenase [235] and the consequences at a molecular level are starting to emerge. Apoptosis-inducing factor is a flavoprotein that can stimu- late a caspase-independent cell-death pathway required for early embryonic morphogenesis [236,237]. To gain further insight into the redox properties of apoptosis-inducing factor, Lys176 and Glu313, located near the isoalloxazine ring of FAD, were individually changed into alanine by site-directed mutagenesis. Both apoptosis-inducing factor variants appeared to be highly active when assayed in the presence of excess FAD. However, during purification the Lys176Ala and Glu313Ala mutant enzymes easily lost the flavin cofactor, yielding the corresponding apoproteins [238]. This again demonstrates that changing a specific amino-acid residue can considerably influence the strength of flavin binding. In the near future, other recombinant-based methods such as the construction of fusion proteins will undoubtedly emerge for flavoprotein deflavination and reconstitution. 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ARTICLE Deflavination and reconstitution of flavoproteins Tackling fold and function Marco H. Hefti*, Jacques Vervoort and Willem J. H. van Berkel Laboratory of. dissociation of flavoproteins into apo- protein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of