Mutational study on alphaGln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771

Nitrile hydratase (NHase) from Rhodococcus sp. N771 is a non-heme iron enzyme having post-translationally modified cysteine ligands, alphaCys112-SO2H and alphaCys114-SOH. We replaced alphaGln90, which is conserved in all known NHases and involved in the hydrogen-bond network around the catalytic center, with glutamic acid or asparagine. The kcat of alphaQ90E and alphaQ90N mutants decreased to 24% and 5% that of wild type respectively, but the effect of mutations on Km was not very significant. In both mutants, the alphaCys114-SOH modification appeared to be responsible for the catalysis as in native NHase. We crystallized the nitrosylated alphaQ90N mutant and determined its structure at a resolution of 1.43 A. The structure was basically identical to that of native nitrosylated NHase except for the mutated site and its vicinity. The structural difference between native and alphaQ90N mutant NHases suggested the importance of the hydrogen bond networks between alphaGln90 and the iron center for the catalytic activity.     

Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy

Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.     

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.     

Post-translational modification is essential for catalytic activity of nitrile hydratase

Nitrile hydratase from Rhodococcus sp. N-771 is an alphabeta heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, alphaCys112 and alphaCys114 are modified to a cysteine sulfinic acid (Cys-SO2H) and a cysteine sulfenic acid (Cys-SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The alphabeta complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC/MS analysis showed that the anaerobically reconstituted alphabeta complex did not have the modification of alphaCys112-SO2H and aerobic incubation induced the modification. The activity of the reconstituted alphabeta complex correlated with the amount of alphaCys112-SO2H. Furthermore, ESI-LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that alphaCys114 is modified to Cys-SOH together with the sulfinic acid modification of alphaCys112. These results suggest that alphaCys112 and alphaCys114 are spontaneously oxidized to Cys-SO2H and Cys-SOH, respectively, and alphaCys112-SO2H is responsible for the catalytic activity solely or in combination with alphaCys114-SOH.     

Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.     

Molecular dynamics simulations of the photoactive protein nitrile hydratase

Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, α and β. Hydrogen bonds between βArg-56 and αCys-114 sulfenic acid (αCEA114) are important to maintain the enzymatic activity. The enzyme may be inactivated by endogenous nitric oxide (NO) and activated by absorption of photons of wavelength λ < 630 nm. To explain the photosensitivity and to propose structural determinants of catalytic activity, differences in the dynamics of light-active and dark-inactive forms of NHase were investigated using molecular dynamics (MD) modeling. To this end, a new set of force field parameters for nonstandard NHase active sites have been developed. The dynamics of the photodissociated NO ligand in the enzyme channel was analyzed using the locally enhanced sampling method, as implemented in the MOIL MD package. A series of 1 ns trajectories of NHases shows that the protonation state of the active site affects the dynamics of the catalytic water and NO ligand close to the metal center. MD simulations support the catalytic mechanism in which a water molecule bound to the metal ion directly attacks the nitrile carbon.     

Cobalt-substituted Fe-type nitrile hydratase of Rhodococcus sp. N-771

When the genes encoding alpha and beta subunits of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 were expressed in Escherichia coli in Co-supplemented medium without co-expression of the NHase activator, the NHase specifically incorporated not Fe but Co ion into the catalytic center. The produced Co-substituted enzyme exhibited rather weak NHase activity, initially. However, the activity gradually increased by the incubation with an oxidizing agent, potassium hexacyanoferrate. The oxidizing agent is likely to activate the Co-substituent by oxidizing the Co atom to a low-spin Co(3+) state and/or modification of alphaCys-112 to a cysteine-sulfinic acid. It is suggested that the NHase activator not only supports the insertion of an Fe ion into the NHase protein but also activates the enzyme via the oxidation of its iron center.     

High resolution X-ray molecular structure of the nitrile hydratase from Rhodococcus erythropolis AJ270 reveals posttranslational oxidation of two cysteines into sulfinic acids and a novel biocatalytic nitrile hydration mechanism

The crystal structure of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 was determined at 1.3A resolution. The two cysteine residues (alphaCys(112) and alphaCys(114)) equatorially coordinated to the ferric ion were post-translationally modified to cysteine sulfinic acids. A glutamine residue (alphaGln(90)) in the active center gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion.     

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe³⁺ or Co³⁺ center with two modified Cys ligands: cysteine sulfininate (Cys-SO₂ ⁻) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO₂ ⁻, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.     

Fe-type nitrile hydratase

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.     

Thermal equilibrium of two conformations in photosensitive nitrile hydratase probed by the FTIR band of nitric oxide bound to the non-heme iron center

Nitrile hydratase (NHase) from Rhodococcus N-771 is a novel enzyme that is inactive in the dark due to an enodogenous nitric oxide (NO) molecule bound to the non-heme iron center, and is activated by its photodissociation. FTIR spectra in the NO stretching region of the dark-inactive NHase were recorded in the temperature range of 270-80 K. Two NO peaks were observed at 1854 and 1846 cm-1 at 270 K, and both frequencies upshifted as the temperature was lowered, retaining the peak separation of 8-9 cm-1. The relative intensity of the lower-frequency peak increased with decreasing temperature up to ~120 K, whereas it was mostly unchanged below this temperature. This observation indicates that two distinct conformations with slightly different NO structures are thermally equilibrated in the dark-inactive NHase above ~120 K, and the interconversion is frozen-in at lower temperatures. The intensity ratio of the NO bands changed gradually upon increasing the pH from 5.5 to 11.0, but no specific pKa value was found. This result, together with the comparison of the light-induced FTIR difference spectra measured at pH 6.5 and 9.0, suggests that the protonation/deprotonation of a specific amino acid group in the active site of NHase is not a direct cause of the occurrence of the two conformations, although several protonatable groups in the protein may influence the energetics of the two conformers. From the previous observation that the isolated alpha subunit of NHase exhibited a single broad NO peak, it is suggested that interaction of the beta subunit forming the reactive cavity is essential for the double-minimum potential of the active-site structure. The frequencies and widths of the two NO bands changed upon addition of propionamide, 1,4-dioxane, and cyclohexyl isocyanide, indicating that these compounds are bound to the active pocket and change the interactions of the iron center or the dielectric environments around the NO molecule. Thus, the NO bands of NHase can also be a useful probe to monitor the binding of substrates and their analogues to the active pocket.     

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.     

Crystal structure of cobalt-containing nitrile hydratase.

The crystal structure of cobalt-containing nitrile hydratase from Pseudonocardia thermophila JCM 3095 at 1.8 A resolution revealed the structure of the noncorrin cobalt at the catalytic center. Two cysteine residues (alphaCys(111) and alphaCys(113)) coordinated to the cobalt were posttranslationally modified to cysteine-sulfinic acid and to cysteine-sulfenic acid, respectively, like in iron-containing nitrile hydratase. A tryptophan residue (betaTrp(72)), which may be involved in substrate binding, replaced the tyrosine residue of iron-containing nitrile hydratase. The difference seems to be responsible for the preference for aromatic nitriles rather than aliphatic ones of cobalt-containing nitrile hydratase.     

Arginine 56 mutation in the beta subunit of nitrile hydratase: importance of hydrogen bonding to the non-heme iron center

Arginine 56 in the β subunit (βArg56) of the iron-containing nitrile hydratase (NHase), one of the strongly conserved residues within the NHase family, is known to form hydrogen bonds to the sulfinyl (–SO₂H) and sulfenyl (–SOH) groups of the post-translationally modified cysteine residues in the catalytic center. βArg56 was substituted by tyrosine, glutamate or lysine, respectively, and the respective mutant enzymes generated by reconstitution were characterized. The βR56K mutant complex exhibited about 1% of the enzymatic activity of native NHase, while the others were totally inactive. The kinetic analysis of the βR56K mutant complex exhibited a drastic decrease in turnover number and decreases in kinetic constants for substrate and inhibitors as compared to the native NHase. Changes in UV–visible absorption and light-induced Fourier transform infrared difference spectra suggest that βArg56 is involved in the positioning of the –SO₂H and –SOH groups of the modified Cys residues in the catalytic center so as to fine tune the electronic state of the iron center suitable for catalysis. Thus, βArg56 is essential for catalysis.     

Nitrile pathway involving acyl-CoA synthetase: overall metabolic gene organization and purification and characterization of the enzyme

Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 +/- 0.04, 0.37 +/- 0.02, and 0.22 +/- 0.02 mm, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) comprising these enzymes.     

Motif CXCC in nitrile hydratase activator is critical for NHase biogenesis in vivo

Nitrile hydratase (NHase) activator from Rhodococcus sp. N-771 is required for NHase functional expression. The motif 73CXCC76 in the NHase activator sequence was here revealed to be vital for its function by site-directed mutagenesis. All three substitutions of the cysteines by serines resulted in a much lower level of expression of active NHase. Furthermore, interaction between NHase activator and NHase was detected and the critical role of NHase activator was not exhibited in the cysteine oxidization process of NHase. These findings suggest NHase activator mainly participates in iron trafficking in NHase biogenesis as an iron type metallochaperone.     

Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt-containing NHase (Co-NHase) was reported to depend on self-subunit swapping and the activator of the Co-NHase acts as a self-subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit-fused Co-NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL-18668 and the activator of low-molecular-mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt-containing proteins. Substitution of the amino acids involved in the C-terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt-binding sites might be located at the flexible C-terminal region. The cobalt-free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit-fused NHases and confirmed the metallochaperone function of the self-subunit swapping chaperone.     

Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.

The crystal structure analysis of the Fe-type nitrile hydratase from Rhodococcus sp. N-771 revealed the unique structure of the enzyme composed of the alpha- and beta-subunits and the unprecedented structure of the non-heme iron active center [Nagashima, S., et al. (1998) Nat. Struct. Biol. 5, 347-351]. A number of hydration water molecules were identified both in the interior and at the exterior of the enzyme. The study presented here investigated the roles of the hydration water molecules in stabilizing the tertiary and the quaternary structures of the enzyme, based on the crystal structure and the results from a laser light scattering experiment for the enzyme in solution. Seventy-six hydration water molecules between the two subunits significantly contribute to the alphabeta heterodimer formation by making up the surface shape, forming extensive networks of hydrogen bonds, and moderating the surface charge of the beta-subunit. In particular, 20 hydration water molecules form the extensive networks of hydrogen bonds stabilizing the unique structure of the active center. The amino acid residues hydrogen-bonded to those hydration water molecules are highly conserved among all known nitrile hydratases and even in the homologous enzyme, thiocyanate hydrolase, suggesting the structural conservation of the water molecules in the NHase family. The crystallographic asymmetric unit contained two heterodimers connected by 50 hydration water molecules. The heterotetramer formation in crystallization was clearly explained by the concentration-dependent aggregation state of NHase found in the light scattering measurement. The measurement proved that the dimer-tetramer equilibrium shifted toward the heterotetramer dominant state in the concentration range of 10(-2)-1.0 mg/mL. In the tetramer dominant state, 50 water molecules likely glue the two heterodimers together as observed in the crystal structure. Because NHase exhibits a high abundance in bacterial cells, the result suggests that the heterotetramer is physiologically relevant. In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center. This finding may give a clue for changing the substrate specificity of the enzyme. Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel. Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light.     

Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.     

X-ray analysis of ferredoxin from Spirulina platensis. II. Chelate structure of active center.

A chloroplast-type ferredoxin from Spirulina platenis crystallized in an orthorhombic system, space group C2221, with cell dimensions a=62.32, b=28.51, and c=108.08 A. The electron density map at 2.8 A resolution was prepared by using the best phase angles determined by the single isomorphous replacement method coupled with the anomalous dispersion method. The chelating structure of the acitve center was revealed as follows. Of the six cysteinyl residues in the molecule, Cys 41, Cys 4k, Cys 49, and Cys 79 are involved in the active center. Cys 41 and Cys 46 are coordinated to one iron atom, and Cys 49 and Cys 79 to the other iron atom. Only one of these cysteinyl residues, Cys 79, is comparatively apart from the other three in the amino acids sequence of the molecule, as found in the case of bacterial ferredoxin. It appears that the NH....S hydrogen bonds are around the active center, as in other non-heme iron sulfur proteins.