Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: nature and possible function of compound ES

Previously, we reported spectroscopic properties of cytochrome P450cam compound I, (ferryl iron plus a porphyrin π-cation radical (Fe^IV = O/Por⁺)), as well as compound ES (Fe^IV = O/Tyr) in reactions of substrate-free ferric enzyme with m-chloroperbenzoic acid [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-9]. Compound ES arises by intramolecular electron transfer from nearby tyrosines to the porphyrin π-cation radical of Compound I, and has been characterized by rapid-freeze-quench-Mössbauer/EPR spectroscopy; the tyrosyl radical was assigned to Tyr96 for wild type or to Tyr75 for the Tyr96Phe variant [V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.L. Barra, S.G. Sligar, A.X. Trautwein, J. Biol. Chem. 279 (2004) 10919–10930]. Here we report rapid-scanning stopped-flow studies of the reactions of peracids with substrate-free ferric Y75F, Y96F, and Y96F/Y75F P450cam variants, showing how these active site changes influence electron transfer from nearby tyrosines and affect formation of intermediates. Curiously, rates of generation of Compounds I and ES for both single mutants were not very different from wild type. Contrasting with the earlier EPR results, the Y96F/Y75F variant was also shown to form an ES-like species, but more slowly. When substrate is not present, or is improperly bound, compound I rapidly converts to compound ES, which can be reduced to form H₂O and ferric P450, thus avoiding the modification of nearby protein groups or release of reactive oxygen species.     

The role of tyrosine 343 in substrate binding and catalysis by human sulfite oxidase

In the crystal structure of chicken sulfite oxidase, the residue Tyr(322) (Tyr(343) in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, k(cat) was somewhat impaired ( approximately 34% wild-type activity at pH 8.5), whereas the K(m)(sulfite) showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, k(red)(heme) changed very little over the entire pH range, with a significant increase in K(d)(sulfite) at high pH. The k(red)(heme) of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k(red)(heme) and K(d)(sulfite) were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (k(red)(Mo) = 972 s(-1) at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr(343) residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.     

Mutational analysis of stress-responsive peanut dual specificity protein kinase. Identification of tyrosine residues involved in regulation of protein kinase activity

We recently reported that Arachis hypogaea serine/threonine/tyrosine (STY) protein kinase is developmentally regulated and is induced by abiotic stresses (Rudrabhatla, P., and Rajasekharan, R. (2002) Plant Physiol. 130, 380-390). Other than MAPKs, the site of tyrosine phosphorylation has not been documented for any plant kinases. To study the role of tyrosines in the phosphorylation of STY protein kinase, four conserved tyrosine residues were sequentially substituted with phenylalanine and expressed as histidine fusion proteins. Mass spectrometry experiments showed that STY protein kinase autophosphorylated within the predicted kinase ATP-binding motif, activation loop, and an additional site in the C terminus. The protein kinase activity was abolished by substitution of Tyr(297) with Phe in the activation loop between subdomains VII and VIII. In addition, replacing Tyr(148) in the ATP-binding motif and Tyr(317) in the C-terminal domain with Phe not only obliterated the ability of the STY protein kinase protein to be phosphorylated, but also inhibited histone phosphorylation, suggesting that STY protein kinase is phosphorylated at multiple sites. Replacing Tyr(213) in the Thr-Glu-Tyr sequence motif with Phe resulted in a 4-fold increase in autophosphorylation and 2.8-fold increase in substrate phosphorylation activities. Mutants Y148F, Y297F, and Y317F displayed dramatically lower phosphorylation efficiency (k(cat)/K(m)) with ATP and histone, whereas mutant Y213F showed increased phosphorylation. Our results suggest that autophosphorylation of Tyr(148), Tyr(213), Tyr(297), and Tyr(317) is important for the regulation of STY protein kinase activity. Our study reveals the first example of Thr-Glu-Tyr domain-mediated autoinhibition of kinases.     

The role of redox-active amino acids on compound I stability, substrate oxidation, and protein cross-linking in yeast cytochrome C peroxidase.

The role of two tryptophans (Trp51 and Trp191) and six tyrosines (Tyr36, Tyr39, Tyr42, Tyr187, Tyr229, and Tyr236) in yeast cytochrome c peroxidase (CcP) has been probed by site-directed mutagenesis. A series of sequential mutations of these redox-active amino acid residues to the corresponding, less oxidizable residues in lignin peroxidase (LiP) resulted in an increasingly more stable compound I, with rate constants for compound I decay decreasing from 57 s(-1) for CcP(MI, W191F) to 7 s(-1) for CcP(MI, W191F,W51F,Y187F,Y229F,Y236F,Y36F,Y39E,Y42F). These results provide experimental support for the proposal that the stability of compound I depends on the number of endogenous oxidizable amino acids in proteins. The higher stability of compound I in the variant proteins also makes it possible to observe its visible absorption spectroscopic features more clearly. The effects of the mutations on oxidation of ferrocytochrome c and 2,6-dimethoxyphenol were also examined. Since the first mutation in the series involved the change of Trp191, a residue that plays a critical role in the electron transfer pathway between CcP and cyt c, the ability to oxidize cyt c was negligible for all mutant proteins. On the other hand, the W191F mutation had little effect on the proteins' ability to oxidize 2,6-dimethoxyphenol. Instead, the W51F mutation resulted in the largest increase in the k(cat)/K(M), from 2.1 x 10(2) to 5.0 x 10(3) M(-1) s(-1), yielding an efficiency that is comparable to that of manganese peroxidase (MnP). The effect in W51F mutation can be attributed to the residue's influence on the stability and thus reactivity of the ferryl oxygen of compound II, whose substrate oxidation is the rate-determining step in the reaction mechanism. Finally, out of all mutant proteins in this study, only the variant containing the Y36F, Y39E, and Y42F mutations was found to prevent covalent protein cross-links in the presence of excess hydrogen peroxide and in the absence of exogenous reductants. This finding marks the first time a CcP variant is incapable of forming protein cross-links and confirms that one of the three tyrosines must be involved in the protein cross-linking.     

Redox control of iodotyrosine deiodinase

The redox chemistry of flavoproteins is often gated by substrate and iodotyrosine deiodinase (IYD) has the additional ability to switch between reaction modes based on the substrate. Association of fluorotyrosine (F‐Tyr), an inert substrate analog, stabilizes single electron transfer reactions of IYD that are not observed in the absence of this ligand. The co‐crystal of F‐Tyr and a T239A variant of human IYD have now been characterized to provide a structural basis for control of its flavin reactivity. Coordination of F‐Tyr in the active site of this IYD closely mimics that of iodotyrosine and only minor perturbations are observed after replacement of an active site Thr with Ala. However, loss of the side chain hydroxyl group removes a key hydrogen bond from flavin and suppresses the formation of its semiquinone intermediate. Even substitution of Thr with Ser decreases the midpoint potential of human IYD between its oxidized and semiquinone forms of flavin by almost 80 mV. This decrease does not adversely affect the kinetics of reductive dehalogenation although an analogous Ala variant exhibits a 6.7‐fold decrease in its k_cat/K_m. Active site ligands lacking the zwitterion of halotyrosine are not able to induce closure of the active site lid that is necessary for promoting single electron transfer and dehalogenation. Under these conditions, a basal two‐electron process dominates catalysis as indicated by preferential reduction of nitrophenol rather than deiodination of iodophenol.     

Molecular Basis for Enzymatic Sulfite Oxidation: HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY*S?

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased K_{m(sulfite)(app)} by 2–3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDH^WT and SDH^Y236F, the catalytic turnover rates of SDH^R55M and SDH^H57A are relatively insensitive to pH (∼60 and 200 s^–1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDH^H57A and SDH^Y236F) or absence of Arg-55 (SDH^R55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDH^R55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDH^R55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.Sulfite-oxidizing enzymes protect cells against potentially fatal damage to DNA and proteins caused by exposure to sulfite, and consequently they are found in all forms of life (1). In bacteria, sulfite oxidation is often linked to energy-generating processes during chemolithotrophic growth on reduced sulfur compounds (2, 3), whereas both plant and vertebrate sulfite oxidases have been shown to detoxify sulfite arising from the degradation of methionine and cysteine and exposure to sulfur dioxide (4, 5).All known sulfite-oxidizing enzymes belong to the same family of mononuclear molybdenum enzymes. Their active sites contain one molybdopterin unit per molybdenum atom, and these enzymes may also contain heme groups as accessory redox centers (6–9). Examples of different types of sulfite-oxidizing molybdoenzymes are the homodimeric plant sulfite oxidase, which does not contain a heme group and uses oxygen as its preferred electron acceptor (9), the homodimeric chicken and human liver sulfite oxidases (CSO³ and HSO, respectively) (10), which are also able to use oxygen as an electron acceptor, and the bacterial sulfite dehydrogenase (SDH) isolated from the soil bacterium Starkeya novella (11, 12), which cannot donate electrons directly to oxygen. Each monomer of CSO and HSO contains a heme b center in addition to the molybdenum center, and the redox centers are located within separate, flexibly linked domains of the same protein subunit. In contrast, the bacterial enzyme is a heterodimer where each subunit of the enzyme contains one redox center. The molybdopterin cofactor is located in the larger 40.2-kDa SorA subunit, and the c-type heme is located in the smaller, 8.8-kDa SorB subunit (12). The SDH quaternary structure thus differs clearly from that of the human and chicken sulfite oxidases.Crystal structures are available for plant sulfite oxidase, CSO, and the bacterial SDH (10, 11, 13–15) and have revealed molecular details of the sulfite-oxidizing enzymes. In the CSO structure, the mobile heme b domain occupies a position too removed from the molybdenum active site to mediate efficient electron transfer (10), and indeed the kinetics of this enzyme are known to be complicated by domain movements (16). In contrast, the bacterial SDH is a tight complex with strong electrostatic interactions between the subunits, and the close approach of the redox centers (Mo–Fe distance 16.6 Å) allows for rapid electron transfer (11, 17) (Fig. 1, A and B).Open in a separate windowFIGURE 1.Details of the crystal structure of wild type SDH and comparison with CSO. A, ribbon diagram of the SDH heterodimer with the SorA and SorB subunits colored blue and cyan, respectively, and the redox cofactors in space-filling mode with the molybdenum atom colored green and the iron atom colored violet. B, ribbon diagram of a single subunit of CSO with the molybdopterin binding domain in the same orientation as SorA in A. The cytochrome domain of CSO is clearly in a different position with respect to the molybdenum cofactor than is seen for the cytochrome subunit of SDH. C, SDH molybdopterin cofactor demonstrating the geometry of the molybdenum ligands. The thiol ligands donated by the organic component of molybdopterin and the Cys-104 side chain, and the reactive oxygen ligand (O_eq) sit in the equatorial plane with the axial oxygen (O_ax) ligand at the apex of a square pyramid. Atoms are colored as follows: molybdenum (green), sulfur (orange), phosphorous (magenta), oxygen (red), nitrogen (blue), and carbon (yellow in the cofactor and white in the protein). D, hydrogen bonding network around the substrate binding site. The molybdopterin and heme cofactors are shown together with active site residues Cys-104, Arg-55, His-57, Tyr-236, and Gln-33. Figs. 1 and ​and44 were prepared using Pymol (37).Despite the overall structural differences of these proteins, the coordination geometries of the molybdenum active sites of these sulfite-oxidizing enzymes are nearly identical. The oxidized molybdenum center has a square pyramidal conformation, with three sulfur and two oxo ligands (18). Within this molybdenum center, the equatorial oxo ligand is proposed to be catalytically active, whereas the axial oxo ligand is not thought to participate directly in the reaction (Fig. 1C). During catalysis, the equatorial oxo ligand is transformed into a hydroxy/water ligand as a result of the reduction of the molybdenum center (Fig. 2), and it is in this form that it is generally observed in the CSO and SDH crystal structures.Open in a separate windowFIGURE 2.Proposed reaction mechanism for S. novella sulfite dehydrogenase. The reaction is shown in terms of the redox states of the molybdenum and heme centers present in the enzyme. Shown in boldface type and boxed are the stable redox states of the S. novella SDH. Cyt. c, a mitochondrial type cytochrome c₅₅₀ (e.g. horse heart or S. novella cytochrome c₅₅₀) that can act as the external electron acceptor.SDH, CSO, and HSO show similarly high affinities for their substrate, sulfite, and several highly conserved residues surround the substrate-binding and molybdenum active site, namely Tyr-236 (all residues given in SDH numbering (11)), Arg-55, and His-57 (Fig. 1D). Both Arg-55 and Tyr-236 form hydrogen bonds to the catalytically active equatorial Mo-oxo group, whereas His-57 is positioned close to both Arg-55 and Tyr-236 (10, 11) (Fig. 1D). In addition, the crystal structure of the bacterial SDH shows that Arg-55 interacts directly with the second SDH redox center by hydrogen bonding to heme propionate-6 (Fig. 1D) (11).As a result of the similarities in catalytic parameters and the structure of the active site, the bacterial SDH is a very good system for studies of enzymatic sulfite oxidation and especially the molecular basis for catalysis. Since this enzyme does not rely on domain movement for catalysis, it has a less complicated reaction mechanism than the vertebrate enzymes, which facilitates the interpretation of kinetic data, and it can be readily crystallized with both redox centers present in an electron transfer competent conformation. We have previously reported data on the structure, kinetics, EPR, and redox properties of a Y236F-substituted SDH (13). In addition to reduced turnover and substrate affinity, this substitution influences the reactivity of the SDH toward oxygen, turning SDH^Y236F essentially into an (albeit weak) sulfite oxidase. In order to further understand the roles of the conserved amino acids surrounding the molybdenum active site of sulfite-oxidizing enzymes, we have created two novel amino acid substitutions in the Arg-55 and His-57 residues present at the active site and have investigated their effect on catalytic and spectroscopic parameters of the bacterial SDH. We have also solved the crystal structures of the substituted enzymes, which have provided new insights into the conformation and plasticity of the active site of sulfite-oxidizing enzymes and how the conserved active site residues contribute to sulfite oxidation.     

Role of conserved tyrosine 343 in intramolecular electron transfer in human sulfite oxidase

Tyrosine 343 in human sulfite oxidase (SO) is conserved in all SOs sequenced to date. Intramolecular electron transfer (IET) rates between reduced heme (Fe(II)) and oxidized molybdenum (Mo(VI)) in the recombinant wild-type and Y343F human SO were measured for the first time by flash photolysis. The IET rate in wild-type human SO at pH 7.4 is about 37% of that in chicken SO with a similar decrease in k(cat). Steady-state kinetic analysis of the Y343F mutant showed an increase in K(m)(sulfite) and a decrease in k(cat) resulting in a 23-fold attenuation in the specificity constant k(cat)/K(m)(sulfite) at the optimum pH value of 8.25. This indicates that Tyr-343 is involved in the binding of the substrate and catalysis within the molybdenum active site. Furthermore, the IET rate constant in the mutant at pH 6.0 is only about one-tenth that of the wild-type enzyme, suggesting that the OH group of Tyr-343 is vital for efficient IET in SO. The pH dependences of IET rate constants in the wild-type and mutant SO are consistent with the previously proposed coupled electron-proton transfer mechanism.     

Kinetic mechanism and identification of the active site tyrosine residue in Enterobacter amnigenus arylsulfate sulfotransferase.

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a sulfate group from a phenyl sulfate ester to a phenolic acceptor. The kinetic mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v versus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, inhibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha-naphthol. These results correspond to a ping pong bi bi mechanism. By site-directed mutagenesis, we substituted each conserved tyrosine residue with phenylalanine. Among the mutants, Y123F showed severely reduced catalytic activity. We conclude that Tyr 123 is an essential active site residue. A mechanistic hypothesis is presented to account for these observations.     

Tyr225 in aspartate aminotransferase: contribution of the hydrogen bond between Tyr225 and coenzyme to the catalytic reaction

Tyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallographic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the epsilon-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single arginine residue is required for the interaction of the electron transferring flavoprotein (ETF) with three of its dehydrogenase partners

The interaction of several dehydrogenases with the electron transferring flavoprotein (ETF) is a crucial step required for the successful transfer of electrons into the electron transport chain. The exact determinants regarding the interaction of ETF with its dehydrogenase partners are still unknown. Chemical modification of ETF with arginine-specific reagents resulted in the loss, to varying degrees, of activity with medium chain acyl-coenzyme A dehydrogenase (MCAD). The kinetic profiles showed the inactivations followed pseudo-first-order kinetics for all reagents used. For activity with MCAD, maximum inactivation of ETF was accomplished by 2,3-butanedione (4% residual activity after 120 min) and it was shown that modification of one arginine residue was responsible for the inactivation. Almost 100% restoration of this ETF activity was achieved upon incubation with free arginine. However, the same 2,3-butanedione modified ETF only possessed decreased activity with dimethylglycine- (DMGDH, 44%) and sarcosine- (SDH, 27%) dehydrogenases unlike the abolition with MCAD. Full protection of ETF from arginine modification by 2,3-butanedione was achieved using substrate-protected DMGDH, MCAD and SDH respectively. Cross-protection studies of ETF with the three dehydrogenases implied use of the same single arginine residue in the binding of all three dehydrogenases. These results lead us to conclude that this single arginine residue is essential in the binding of the ETF to MCAD, but only contributes partially to the binding of ETF to SDH and DMGDH and thus, the determinants of the dehydrogenase binding sites overlap but are not identical.     

Electron transfer chain reaction of the extracellular flavocytochrome cellobiose dehydrogenase from the basidiomycete Phanerochaete chrysosporium

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome containing flavin and b-type heme, and plays a key role in cellulose degradation by filamentous fungi. To investigate intermolecular electron transfer from CDH to cytochrome c, Phe166, which is located in the cytochrome domain and approaches one of propionates of heme, was mutated to Tyr, and the thermodynamic and kinetic properties of the mutant (F166Y) were compared with those of the wild-type (WT) enzyme. The mid-point potential of heme in F166Y was measured by cyclic voltammetry, and was estimated to be 25 mV lower than that of WT at pH 4.0. Although presteady-state reduction of flavin was not affected by the mutation, the rate of subsequent electron transfer from flavin to heme was halved in F166Y. When WT or F166Y was reduced with cellobiose and then mixed with cytochrome c, heme re-oxidation and cytochrome c reduction occurred synchronously, suggesting that the initial electron is transferred from reduced heme to cytochrome c. Moreover, in both enzymes the observed rate of the initial phase of cytochrome c reduction was concentration dependent, whereas the second phase of cytochrome c reduction was dependent on the rate of electron transfer from flavin to heme, but not on the cytochrome c concentration. In addition, the electron transfer rate from flavin to heme was identical to the steady-state reduction rate of cytochrome c in both WT and F166Y. These results clearly indicate that the first and second electrons of two-electron-reduced CDH are both transferred via heme, and that the redox reaction of CDH involves an electron-transfer chain mechanism in cytochrome c reduction.     

Identification of Tyr74 and Tyr177 as substrate oxidation sites in cationic cell wall-bound peroxidase from Populus alba L

Cationic cell wall-bound peroxidase (CWPO-C) has the capability to oxidize sinapyl alcohol, ferrocytochrome c, and synthetic lignin polymers, unlike most peroxidases that have been characterized in flowering plants, such as horseradish peroxidase and Arabidopsis thaliana peroxidase A2. It has been suggested that the oxidation site is located on the CWPO-C surface, and homology modeling and chemically modified CWPO-C studies suggest that Tyr74 and/or Tyr177 are possible participants in the catalytic site. The present study clarifies the importance of these Tyr residues for substrate oxidation, using recombinant CWPO-C and recombinant mutant CWPO-C with phenylalanine substitution(s) for tyrosine. Such recombinant proteins, produced in Escherichia coli as inclusion bodies, were successfully refolded to yield the active form, and purified recombinant protein solutions exhibited typical spectra of high-spin ferric protein and displayed H(2) O(2) -dependent oxidation of guaiacol, 2,6-dimethoxyphenol, and syringaldazine. Measurement of peroxidase activity with these guaiacyl and syringyl compounds as reducing substrates indicated that a single mutation, Y74F or Y177F, resulted in substantial loss of oxidation activity (～ 40-60% and 82%, respectively). Also, over 95% of the oxidation activity was lost with a double mutation, Y74F/Y177F. These results indicated that Tyr74 and Tyr177, rather than the heme pocket, play a central role in the oxidation of these substrates. This is the first report of active residues on an enzyme surface being identified in a plant peroxidase. This study also suggests that sinapyl alcohol incorporation into lignin is performed by a peroxidase that generates Tyr radicals on its surface.     

New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation.

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.     

Direct catalytic electrochemistry of sulfite dehydrogenase: Mechanistic insights and contrasts with related Mo enzymes

Under hydrodynamic electrochemical conditions with slow cyclic voltammetry sweep rates we have been able to probe catalytic events at the molybdenum active site of sulfite dehydrogenase (SDH) from Starkeya novella adsorbed on an edge plane graphite electrode within a polylysine film. The electrochemically driven catalytic behaviour of SDH mirrors that seen in solution assays suggesting that the adsorbed enzyme retains its native activity. However, at high sulfite concentrations, the voltammetric waveform transforms from the expected sigmoidal profile to a peak-shaped response, similar to that reported for the molybdenum enzymes DMSO reductase and nitrate reductase (NarGHI and NapAB) where a redox reaction at the active site has been associated with a switch to lower activity at high overpotentials. This is the first time a similar phenomenon has been observed in a Mo-containing oxidase/dehydrogenase, which raises a number of interesting mechanistic problems. The potential at which the activity of SDH becomes attenuated only emerges at saturating substrate conditions and occurs at a potential (ca. + 320mV vs NHE) well removed from any known redox couple in the enzyme. These results cannot be explained by the same mechanism adopted for DMSO reductase and nitrate reductase catalysis.     

The catalytic role of tyrosine 254 in flavocytochrome b2 (L-lactate dehydrogenase from baker's yeast). Comparison between the Y254F and Y254L mutant proteins.

Flavocytochrome b2 catalyses the oxidation of L-lactate to pyruvate in yeast mitochondrial intermembrane space. Its flavoprotein domain is a member of a family of FMN-dependent 2-hydroxy-acid-oxidizing enzymes. Numerous solution studies suggest that the first step of the reaction consists of proton abstraction from lactate C2, leading to a carbanion that subsequently yields electrons to FMN. The crystal structure suggests that the enzyme base is His373, and that Tyr254 may be hydrogen bonded to the substrate hydroxyl. Studies carried out with the Y254F mutant [Dubois, J., Chapman, S.K., Mathews, F.S., Reid, G.A. & Lederer, F. (1990) Biochemistry 29, 6393-6400] showed that Tyr254 does not act as a base but stabilizes the transition state. As the mutation did not induce any change in substrate affinity, the question of the existence of the hydrogen bond in the Michaelis complex remained open. Similar results with glycolate oxidase, mutated at the same position, led to the suggestion that these enzymes actually operate via a hydride transfer mechanism [Macheroux, P., Kieweg, V., Massey, V., Soderlind, E., Stenberg, K. & Lindqvist, Y. (1993) Eur. J. Biochem. 213, 1047-1054]. In the present work, we have re-investigated the matter by analysing the properties of a Y254L mutant flavocytochrome b2, as well as the behaviour of the Y254F enzyme with two substrates other than lactate, and a series of inhibitors. The Y254L protein is less efficient with L-lactate than the wild-type enzyme by a factor of 500, but the substrate affinity is unchanged. In contrast, L-phenyllactate and mandelate, poor substrates (the latter acting more as an inhibitor), exhibit an increased affinity. In addition, the Y254L mutant enzyme is more efficient with phenyllactate than lactate as a substrate. In order to rationalize these observations, we have modelled phenyllactate and mandelate in the active site, using previously described modelling experiments with lactate as a starting point. The results indicate that mandelate cannot bind in an orientation allowing proton abstraction by His373, due to steric interference by the side chains of Ala198 and Leu230. It might possibly adopt a binding mode as proposed previously for lactate, which leads to a hydride transfer and with which the 198 and 230 side chains do not interfere. However, other researchers [Sinclair, R., Reid, G.A. & Chapman, S.K. (1998) Biochem. J. 333, 117-120] showed that A198G, L230A and A198G/L230A mutant enzymes exhibit a strongly improved mandelate dehydrogenase activity. These results indicate that relief of the steric crowding facilitates catalysis by enabling a better mandelate orientation at the active site, suggesting that its productive binding mode is similar to that proposed for lactate in the carbanion mechanism. The modelling studies therefore support the hypothesis of a carbanion mechanism for all substrates. In addition, we present the effect of the two mutations at position 254 on the binding of a number of competitive inhibitors (such as sulfite, D-lactate, propionate) and of inhibitors that are known to bind at the active site both when the flavin is oxidized and when it is in the semiquinone state (propionate, oxalate and L-lactate at high concentrations). Unexpectedly, the results indicate that the integrity of Tyr254 is necessary for the binding of these inhibitors at the semiquinone stage.     

Implication by site-directed mutagenesis of Arg314 and Tyr316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase

Sequence alignment of pig mitochondrial NADP-dependent isocitrate dehydrogenase with eukaryotic (human, rat, and yeast) and Escherichia coli isocitrate dehydrogenases reveals that Tyr316 is completely conserved and is equivalent to the E. coli Tyr345, which interacts with the 2'-phosphate of NADP in the crystal structure [Hurley et al., Biochemistry 30 (1991) 8671-8678]. Lys321 is also completely conserved in the five isocitrate dehydrogenases. Either an arginine or lysine residue is found among the enzymes from other species at the position corresponding to the pig enzyme Arg314. While Arg323 is not conserved among all species, its proximity to the coenzyme site makes it a good candidate for investigation. The importance of these four amino acids to the function of pig mitochondrial NADP-isocitrate dehydrogenase was studied by site-directed mutagenesis. Mutants (R314Q, Y316F, Y316L, K321Q, and R323Q) were generated by a megaprimer polymerase chain reaction method. Wild-type and mutant enzymes were expressed in E. coli and purified to homogeneity. All mutant and wild-type enzymes exhibited comparable molecular weights indicative of the dimeric enzyme. Mutations do not cause an appreciable change in enzyme secondary structure as revealed by circular dichroism measurements. The kinetic parameters (V(max) and K(M) values) of K321Q and R323Q are similar to those of wild-type, indicating that Lys321 and Arg323 are not involved in enzyme function. R314Q exhibits a 10-fold increase in K(M) for NADP as compared to that of wild-type, while they have comparable V(max) values. These results suggest that Arg314 contributes to the affinity between the enzyme and NADP. The hydroxyl group of Tyr316 is not required for enzyme function since Y316F exhibits similar kinetic parameters to those of wild-type. Y316L shows a 4-fold increase in K(M) for NADP and a decrease in V(max) as compared to wild-type, suggesting that the aromatic ring of the Tyr of isocitrate dehydrogenase contributes to the affinity for coenzyme, as well as to catalysis. The K(i) for NAD of R314Q, Y316F, and Y316L is comparable to that of wild-type, indicating that the Arg314 and Tyr316 may be located near the 2'-phosphate of enzyme-bound NADP.     

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated.We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo^VI/V potential lowered by ca. 60–80 mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT^WT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K_Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.     

Flavinylation and Assembly of Succinate Dehydrogenase Are Dependent on the C-terminal Tail of the Flavoprotein Subunit

The enzymatic function of succinate dehydrogenase (SDH) is dependent on covalent attachment of FAD on the ∼70-kDa flavoprotein subunit Sdh1. We show presently that flavinylation of the Sdh1 subunit of succinate dehydrogenase is dependent on a set of two spatially close C-terminal arginine residues that are distant from the FAD binding site. Mutation of Arg⁵⁸² in yeast Sdh1 precludes flavinylation as well as assembly of the tetrameric enzyme complex. Mutation of Arg⁶³⁸ compromises SDH function only when present in combination with a Cys⁶³⁰ substitution. Mutations of either Arg⁵⁸² or Arg⁶³⁸/Cys⁶³⁰ do not markedly destabilize the Sdh1 polypeptide; however, the steady-state level of Sdh5 is markedly attenuated in the Sdh1 mutant cells. With each mutant Sdh1, second-site Sdh1 suppressor mutations were recovered in Sdh1 permitting flavinylation, stabilization of Sdh5 and SDH tetramer assembly. SDH assembly appears to require FAD binding but not necessarily covalent FAD attachment. The Arg residues may be important not only for Sdh5 association but also in the recruitment and/or guidance of FAD and or succinate to the substrate site for the flavinylation reaction. The impaired assembly of SDH with the C-terminal Sdh1 mutants suggests that FAD binding is important to stabilize the Sdh1 conformation enabling association with Sdh2 and the membrane anchor subunits.     

Studies of the Mo(V) Center of the Y343F Mutant of Human Sulfite Oxidase by Variable Frequency Pulsed EPR Spectroscopy

The Mo(V) forms of the Tyr343Phe (Y343F) mutant of human sulfite oxidase (SO) have been investigated by continuous wave (CW) and variable frequency pulsed EPR spectroscopies as a function of pH. The CW EPR spectrum recorded at low-pH (∼6.9) has g-values similar to those known for the low-pH form of the native vertebrate SO (original lpH form); however, unlike the spectrum of original lpH SO, it does not show any hyperfine splittings from a nearby exchangeable proton. The detailed electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) experiments also did not reveal any nearby protons that could belong to an exchangeable ligand at the molybdenum center. These results suggest that under low-pH conditions the active site of Y343F SO is in the “blocked” form, with the Mo(V) center coordinated by sulfate. With increasing pH the EPR signal from the “blocked” form decreases, while a signal similar to that of the original lpH form appears and becomes the dominant signal at pH >9. In addition, both the CW EPR and ESE-detected field-sweep spectra reveal a considerable contribution from a signal similar to that usually detected for the high-pH form of native vertebrate SO (original hpH form). The nearby exchangeable protons in both of the component forms observed at high-pH were studied by the ESEEM spectroscopy. These results indicate that the Y343F mutation increases the apparent pK_a of the transition from the lpH to hpH forms by ∼2 pH units.     

Sulfite oxidizing enzymes

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.