Soret Spectral and Bioinformatic Approaches Provide Evidence for a Critical Role of the α -Subunit in Assembly of Tetrameric Hemoglobin

Soret spectral contributions of the α-subunit heme pocket have been evaluated by performing static titrations of apohemoglobin A with CNProtohemin under varied experimental conditions. Increasing the temperature from 5 to 30°C in 0.05 M potassium phosphate buffer, pH 7, resulted in a decreasingly prominent hypsochromic shifts reflecting altered the vinyl–globin interactions. Studies at 10°C in over pH range of 6.7–8.0 revealed a profile for the spectral shifts approximating the side chain pK value (7.4) a histidyl residue. These overall spectral changes correspond to ΔE of ≤7 kJ/mol indicative of electrostatic noncovalent interactions. Further our current molecular modeling studies indicate that the spatial arrangement and critical noncovalent interactions of tyrosine 42 and histidine 45 (aromatic residues unique to the α-subunit) make significant contribution to the Soret spectra. Most interestingly, phylogenetic analyses have revealed the presence of a histidyl triad in the α-chain of all vertebrates that form heterotetramers.     

Esterification of the propionate groups promotes alpha/beta hemoglobin chain homogeneity of CN-hemin binding

This study examines the post-translational role of peripheral propionate groups in the incorporation of the Fe-protoporphryin IX heme into nascent alpha- and beta-globin chains. Human apohemoglobin (a heme-free alpha/beta dimer) in 0.05 M potassium phosphate buffer, pH 7, at 20 degrees C was titrated with either CN-protohemin (native heme with two peripheral propionate groups), or CN-dimethylester hemin (a modified heme with two methyl ester groups in place of the propionate groups). Soret spectrophotometric CN-hemin titrations confirmed that a spectral shift resulted upon binding of protohemin, but no spectral shift occurred upon binding the dimethylester derivative. Recent studies have correlated a Soret spectral shift with the preferential heme binding to the alpha subunit of apohemoglobin. The absence of a Soret wavelength shift (in conjunction with molecular modeling) presented here suggested that the modification of heme propionate groups prevented the formation of an alpha-heme/beta-globin intermediate, a requisite step in the normal assembly of functional hemoglobin.     

Linking conformation change to hemoglobin activation via chain-selective time-resolved resonance Raman spectroscopy of protoheme/mesoheme hybrids

Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the α and β chains are selectively substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and the yields are the same for the two chains, consistent with recent results on ¹⁵N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme ν ₄ and ν _Fe–His RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UV RR spectra. Both chains show Fe–His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, R_deoxy (0.07 μs), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe–His bond weakens, more so for the α chains than for the β chains. The weakening is gradual for the β chains, but is abrupt for the α chains, coinciding with completion of the R–T quaternary transition, at 20 μs. Since the transition from fast- to slow-rebinding Hb also occurs at 20 μs, the drop in the α chain ν _Fe–His supports the localization of ligation restraint to tension in the Fe–His bond, at least in the α chains. The mechanism is more complex in the β chains.     

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

 The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. Received: 17 July 1996 / Accepted: 28 November 1996     

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV–vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k ₁ but a larger k ₂. Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.     

Inhibition of aromatase cytochrome P-450 by 10-oxirane and 10-thiirane substituted androgens. Implications for the structure of the active site

The mechanism of inhibition of estrogen synthetase (P-450arom) by 19R- and 19S-isomers of 10-oxiranyl-and 10-thiiranyl-4-estrene-3,17-dione was investigated using human placental microsomes and purified enzyme preparations. The 19R-isomers were potent inhibitors and exhibited affinities 36-fold (10-oxirane) and 80-fold (10-thiirane) greater than the respective 19S-isomers. Kinetic experiments showed that inhibition by the 19R-isomers is competitive with respect to substrate; inhibition constants for the (19R)-10-oxirane (Ki = 10 nM) and the 19R-10-thiirane (Ki = 2 nM) indicate that each binds with greater affinity than the androgen substrates androstenedione and testosterone. Inhibition time courses and kinetic data were consistent with high affinity, reversible binding. Spectral titrations of microsomal preparations and purified P-450arom showed that binding of the 19R-isomers shifts the Soret maximum of the ferric enzyme to 411 nm (10-oxirane) or 425 nm (10-thiirane); addition of excess androstenedione reversed the spectral changes, producing the high spin form of the enzyme with a Soret peak at 393 nm. These spectral shifts suggest that the oxygen atom of the 10-oxirane and the sulfur atom of the 10-thiirane are bound to the heme iron in the inhibitor complexes. These results suggest that the high affinities of the inhibitors arise from their dual interaction with the androgen binding site and with the heme. Coordination of the C19 heteroatom to the heme indicates that C19 of androgen substrates may be positioned sufficiently close to the heme to allow direct attack by an iron-bound oxidant. Stereoselective binding of the 19R-isomers by P-450arom further suggests that the heme is likely to be positioned above C1 and C2 of the A ring.     

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin.

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NH's of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.     

The effect of axial ligand plane orientation on the contact and pseudocontact shifts of low-spin ferriheme proteins

 The effect of axial ligand nodal plane orientation on the contact and pseudocontact shifts of a symmetrical low-spin octamethylferriheme center has been calculated as a function of the angle of the axial ligand. Simple Hückel techniques have been used to estimate the contact contribution, and values obtained from model hemes, together with counter-rotation of the g-tensor, have been used to estimate the pseudocontact contribution, for the eight β-pyrrole methyl and four meso-H positions. It is found that the maximum and minimum contact shifts occur when the axial ligand is aligned at an angle of ±15° to the meso-H axes of the heme, rather than when the axial ligand plane lies along the porphyrin nitrogens, as assumed previously by some investigators. For systems having one planar axial ligand or two ligands in parallel planes, the contact and pseudocontact contributions at the meso-H positions are comparable in size (at least on the basis of simple Hückel estimates), while the contact contribution clearly dominates the isotropic shifts of the heme methyls. Allowing for the substituent effect of the 2,4-vinyls of protohemin, or the 2,4-thioethers of hemin c, as well as the average diamagnetic shifts of the heme methyls and meso-H, plots of the predicted shifts as a function of axial ligand nodal plane orientation have been constructed for hemin b- and c-containing proteins. Excellent agreement in the order of shifts, and reasonable agreement in the sizes of the observed shifts, is observed in the majority of the ferriheme proteins for which the methyl and meso-H resonances have been assigned and proton shifts reported. Plots have also been constructed for hemin c-containing proteins having the two axial ligand nodal planes oriented at relative angles of 40°, 70°, and 80°. Excellent agreement in the order of shifts, and reasonable agreement in the magnitudes of the observed shifts, is observed in all cases of bacterial cytochromes which do not fit the plots that assume the ligands are in parallel planes, except one – the cytochrome c-552 of Nitrosomonas europae. Except for this case, where the order of the predicted methyl shifts at any angle of the axial ligands disagrees with the observed, the reasons can usually be attributed to a large dihedral angle between two axial ligand nodal planes, to strong H-bonding interactions involving His and/or CN^– ligands, or to off-axis binding of one (or both) axial ligand(s). Ruffling of the porphyrin ring may also contribute to the contact shift in as yet undefined ways. Hence, despite the simplicity of the calculations, the agreement with observed data is highly satisfying and the concept of the importance of axial ligand plane orientation on the observed proton shifts of heme proteins is fully confirmed. Received: 15 June 1998 / Accepted: 6 August 1998     

Effects of mutations in the WD40 domain of α-COP on its interaction with the COPI coatomer in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Replacement of glycine 227 in the fifth WD40 motif of α-COP/Ret1p/Soo1p by charged or aromatic amino acids is responsible for the temperature-dependent osmo-sensitivity of Saccharomyces cerevisiae, while truncations of WD40 motifs exerted a reduction in cell growth rate and impairment in assembly of cell-wall associated proteins such as enolase and Gas1p. Yeast two-hybrid analysis revealed that the ret1-1/soo1-1 mutation of α-COP abolished the interaction with β- and ɛ-COP, respectively, and that the interaction between α-COP and β-COP relied on the WD40 domain of α-COP. Furthermore, although the WD40 domain is dispensable for interaction of α-COP with ɛ-COP, structural alterations in the WD40 domain could impair the interaction.     

Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as α-mannosylglycerate (α-MG) and α-mannosylglyceramide (α-MGA), whereas R. obamensis accumulated only α-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures, R. marinus responded to water stress by accumulation of α-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, α-mannosylglycerate was the major compatible solute and α-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of α-mannosylglyceramide over α-mannosylglycerate. The absence of α-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of α-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed. Received: February 10, 1998 / Accepted: January 11, 1999     

Synthesis of disaccharide neu5Gcα(2–6)galNAcα as a spacer glycoside

The first synthesis of the Neu5Gc analogue of SiaT _n disaccharide, which can be detected in breast tumors by immunochemical methods, is reported. The regioselective sialylation of (3-trifluoroacetamidopropyl)-2-azido-2-deoxy-α-D-galactopyranoside with peracetate of the methyl ester ofN-acetoxyacetyl-neuraminic acid β-ethylthioglycoside in the presence ofN-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) resulted in the derivatives of α- and β-sialyl(2→6)galactosaminide in 39 and 32% yields, respectively. The catalytic hydrogenolysis of the azido group and subsequentN- andO-acetylation of the α-anomer gave the peracetate of trifluoroacetamidopropyl glycoside. Removal of the protective groups led to glycoside Neu5Gcα2→6GalNAcα-O(CH₂)₃NH₂. Using the Neu5Gc derivative with acetoxyacetyl groups at positions O9 and O4 as a donor increases the α-selectivity of sialylation to afford the α- and β-anomers in 69 and 8% yields, respectively.     

Thermal denaturation of cytochrome c peroxidase: pH dependence

Upon heating cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) at pH 4 and 5, the enzyme precipitates at 41 degrees C and 51 degrees C, respectively. Incubating the enzyme at lower temperatures causes a slow dissociation of the heme from the protein. The heme precipitates, while the apoprotein remains soluble. Between pH 6 and 8, the native enzyme is converted to a low-spin ferric form upon heating. The Soret maximum shifts from 408 to 414 nm. The midpoint of this transition is pH-dependent, with a value of 46 degrees C at pH 6 decreasing to 29 degrees C at pH 8. At high temperatures the 414 nm form is converted to a species which has a 'free heme' spectrum with low absorptivity and Soret maximum at 390 nm. The midpoint temperature of this latter transition is 62 degrees C and 57 degrees C at pH 7 and 8, respectively.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

A Thermostable α-Galactosidase from Lactobacillus fermentum CRL722: Genetic Characterization and Main Properties

α-Galactosidase (α-Gal) enzyme, which is encoded by the melA gene hydrolyzes α-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These α-galacto-oligosaccharides (α-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses α-Gal in the gut. The use of microbial α-Gal is a promising alternative to eliminate α-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction–based techniques, and showed strong similarities with the α-Gal gene of thermophilic microorganisms. The α-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55°C) and stable at wide ranges of temperatures and pH. The thermostable α-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of α-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade α-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value.     

Isolation and characterization of temperature-sensitive mutations in the gene ( rpb3? ) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the α subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β′ subunits. Previously we demonstrated that the Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, as in the case of the prokaryotic α₂β complex. In order to obtain further insight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each (with the exception of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A–D) that are conserved between the homologues of eukaryotic subunit 3. The three Cs mutations were all located in region A, in agreement with the central role of the corresponding region in the assembly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were found in all four regions. Growth of the Ts mutants was reduced to various extents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in polymerase assembly or the mutant RNA polymerases containing mutant Rpb3 subunits are unstable. In accordance with this prediction, the Ts phenotype of all the mutants was suppressed to varying extents by over-expression of Rpb11, the pairing partner of Rpb3 in the core subassembly. We conclude that the majority of rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered. Received: 25 January 1999 / Accepted: 27 April 1999     

Structural characterization of a carbon monoxide adduct of a heme–DNA complex

Abstract  

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3′-terminal G-quartet of the DNA though a π–π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π–π stacking interaction between the pseudo-C ₂-symmetric heme and the C ₄-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C ₂ axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe–CO bond in the complex is similar to that of the Fe–CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.     

Purification and characterization of tubulin from the catfishHeteropneustes fossilis

Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.     

Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography–mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.     

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading α-ketoglutarate dioxygenase

 The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the α-ketoglutarate (α-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and α-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g–tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of α-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding. Received: 3 July 1998 / Accepted: 13 October 1998