Biomedical subjects

D B Goodin

Publications and source records attributed to D B Goodin.

30 records · Page 2Linked to original sources

PubMed1994

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.

Crystallography, X-Ray↗

PubMed1994

Small molecule binding to an artificially created cavity at the active site of cytochrome c peroxidase.

In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.

Amino Acid Sequence↗

PubMed1993

Enhanced oxidation of aniline derivatives by two mutants of cytochrome c peroxidase at tryptophan 51.

Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze the enhanced oxidation of a number of substituted anilines. The reaction of CCP compound ES with mesidine is biphasic, while similar reactions using compound II give monophasic kinetics. These data, in addition to the ratio of the Fe4+ = O and free-radical species observed during steady-state turnover, indicate that reduction of the Trp-191 free radical of compound ES is more rapid than the reduction of the Fe4+ = O species. Transient kinetics were examined for the oxidation of eight mono-substituted anilines by CCP, W51F, and W51A. Each of the aniline derivatives were oxidized by the mutants at rates that exceeded that of the wild-type enzyme, and the rate constant for m-chloroaniline was 400-fold faster for W51F than for wild-type CCP. Variations in the rate constants for the different substrates follow a linear free-energy relationship using the Hammet substituent effect parameter sigma +, implicating electron transfer from the aniline ring in the transition state. For aniline oxidation, the free energy of activation is 3 kcal/mol lower for the mutants than for wild-type CCP, and this is due primarily to an increase in the activation entropy. These results indicate that the enhanced kinetics of W51F and W51A result from a generalized increase in enzyme reactivity characterized by an exo-entropic transition state such as dissociation of bound H2O from the Fe4+ = O center.

Amino Acid Sequence↗

PubMed1993

Comprehensive explanation of the anomalous EPR spectra of wild-type and mutant cytochrome c peroxidase compound ES.

Although the cytochrome c peroxidase/H2O2 reaction product, compound ES, has been a long-standing subject of research, only recently has its broad EPR signal been proven to arise from a radical at Trp-191. Despite this advance, no model has satisfactorily explained the anomalous breadth and shape of this signal, which is conventionally interpreted as having axial symmetry with g parallel approximately 2.04 > g perpendicular approximately 2.01, contrary to expectations for a planar pi radical. Furthermore, these g values exhibit marked temperature and preparation dependencies as well as an unexplained high-field "tail" extending from the g = 2.01 peak. We have reexamined the EPR and ENDOR spectra of compound ES at 35 GHz, as well as those of compound ES in the mutant D235E. This mutation significantly alters the line shape of the Trp-191 free radical. We present a comprehensive model that successfully accounts for the properties of this unusual protein free radical. We show that the EPR spectra of both proteins can be described in terms of a weak exchange interaction between the S = 1 oxyferryl (Fe = O)2+ moiety and a radical on Trp-191; a distribution in protein conformation leads to a distribution in the coupling, which ranges from ferromagnetic to antiferromagnetic. We also derive, for the first time, explicit expressions for frozen-solution and single-crystal spectra of such spin-coupled systems and show that the model accounts for all the data that previously led to apparent anomalies in the interpretation of the frozen-solution and single-crystal [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 349-355] EPR properties. Finally, we have used the CW EPR and pulsed-EPR saturation-recovery methodology to address reports that the broad signal from the spin-coupled Trp-191 radical is accompanied by a minority (approximately 10%), narrow signal that is associated with a radical site other than Trp-191. We find no evidence for such a species and discuss the earlier reports in light of our model.

Amino Acid Sequence↗

PubMed1993

The Asp-His-Fe triad of cytochrome c peroxidase controls the reduction potential, electronic structure, and coupling of the tryptophan free radical to the heme.

The buried charge of Asp-235 in cytochrome c peroxidase (CCP) forms an important hydrogen bond to the histidine ligand of the heme iron. The Asp-His-metal interaction, which is similar to the catalytic triad of serine proteases, is found at the active site of many metalloenzymes and is believed to modulate the character of histidine as a metal ligand. We have examined the influence of this interaction in CCP on the function, redox properties, and iron zero-field splitting in the native ferric state and its effect on the Trp-191 free radical site in the oxidized ES complex. Unlike D235A and D235N, the mutation D235E introduces very little perturbation in the X-ray crystal structure of the enzyme active site, with only minor changes in the geometry of the carboxylate-histidine interaction and no observable change at the Trp-191 free radical site. More significant effects are observed in the position of the helix containing residue Glu-235. However, the small change in hydrogen bond geometry is all that is necessary to (1) increase the reduction potential by 70 mV, (2) alter the anisotropy of the Trp-191 free radical EPR, (3) affect the activity and spin-state equilibrium, and (4) reduce the strength of the iron ligand field as measured by the zero-field splitting. The changes in the redox potential with substitution are correlated with the observed zero-field splitting, suggesting that redox control is exerted through the heme ligand by a combination of electrostatic and ligand field effects. The replacement of Asp-235 with Glu appears to result in a significantly weaker hydrogen bond in which the proton resides essentially with His-175. This hydrogen bond is nevertheless strong enough to prevent the reorientation of Trp-191 and the conversion to one of two low-spin states observed for D235A and D235N. The Asp-His-Fe interaction is therefore as important in defining the redox properties and imidazolate character of His-175 as has been proposed, yet its most important role in peroxidase function may be to correctly orient Trp-191 for efficient coupling of the free radical to the heme and to maintain a high-spin 5-coordinate heme center.

Aspartic Acid↗

PubMed1991

Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1989

Identification by ENDOR of Trp191 as the free-radical site in cytochrome c peroxidase compound ES.

The chemical identity of the amino acid free-radical site that represents one of the two oxidizing equivalents stored in the H2O2-oxidized intermediate (compound ES) of the mitochondrial heme enzyme, cytochrome c peroxidase (CcP) has been sought for almost a quarter of a century. Site-directed mutagenesis alone cannot yield this answer. Low-temperature 35-gigahertz (Q-band) electron nuclear double resonance (ENDOR) spectroscopy was used to examine compound ES prepared from proteins containing specifically deuterated methionine or tryptophan, as well as the amino acid replacement Trp51----Phe. The results definitely identify the site of the radical in compound ES as tryptophan, most likely Trp191.

Binding Sites↗

PubMed1988

A simple and efficient procedure for generating random point mutations and for codon replacements using mixed oligodeoxynucleotides.

A very simple and highly efficient procedure for the generation of single and multiple substitutions in segments of DNA is described which has no requirements for conveniently placed restriction sites, and allows all DNA sequences to be equally accessible. A mixed pool of oligodeoxynucleotides is synthesized by contaminating the monomeric nucleotides with low levels of the other three nucleotides such that the full-length oligonucleotide contains on the average one to two changes per molecule. This pool is used in priming in vitro synthesis of the complementary strand of cloned DNA fragments in M13 or pEMBL vectors which have previously been passed through a dut-, ung- Escherichia coli host. Strong selection for the newly synthesized strand is provided by transforming the heteroduplex into a dut+, ung+ host. Single and multiple substitutions in the carboxy-terminal coding region of the MATa1 gene of Saccharomyces cerevisiae are introduced at high efficiency (25-55%) and the changes are identified by direct sequencing alone. The same principle can be used to generate multiple sets of changes at any specified codon.

Codon↗

PubMed1988

A rapid droplet method for Sanger dideoxy sequencing.

A method for performing the dideoxy sequence reaction on petri dishes is described. It allows rapid manipulation of clones and provides large amounts of sequence information quickly and without the need for elaborate laboratory equipment.

Base Sequence↗

PubMed1987

The peroxide complex of yeast cytochrome c peroxidase contains two distinct radical species, neither of which resides at methionine 172 or tryptophan 51.

The nature of the free radical species observed in the peroxide complex of yeast cytochrome c peroxidase is described for protein variants containing amino acid substitutions at Met-172 and Trp-51. As was the case with Met-172 mutations (Goodin, D.B., Mauk, A.G., and Smith, M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1295-1299), Trp-51 can be substituted to give active enzyme. Phe-51-containing enzyme has a higher turnover rate than the original enzyme and exhibits an altered pH dependence. The properties of the isotropic and axial components (Hoffman, B.M., Roberts, J.E., Kang, C.H., and Margoliash, E. (1981) J. Biol. Chem. 256, 6556-6564; Hori, H., and Yonetani, T. (1985) J. Biol. Chem. 260, 349-355) of the EPR signal of the wild-type enzyme-peroxide complex, studied as a function of H2O2 stoichiometry, support proposals (Goodin et al. (1985) and Hori and Yonetani (1985), see above) that two distinct radical species are formed, and spin quantification shows that the isotropic radical is always formed in substoichiometric amounts. The peroxide complexes for proteins containing amino acid substitutions at either Met-172 or Trp-51 exhibit somewhat larger than normal levels of the isotropic radical signal. In addition, these mutants are unlike wild-type enzyme in that the axial EPR signal associated with the peroxide complex is seen only at 10 K and not at 90 K. Thus, neither amino acid can be considered to be the molecular species responsible for either radical signal, but both mutations appear to affect the physical properties of the axial signal representing the major radical species.

Cloning, Molecular↗

PubMed1986

Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis.

Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4+ = O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild-type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free-radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex.

Amino Acid Sequence↗

PubMedDate not supplied

Investigating resins for solid phase organic synthesis: the relationship between swelling and microenvironment as probed by EPR and fluorescence spectroscopy.

The relationship between observed swelling of two cross-linked polystyrene resins and the microenvironment within polymer matrixes has been examined. Polystyrene cross-linked with either divinyl benzene (Merrifield resin) or 1,4-bis(4-vinylphenoxy)butane (JandaJel) was investigated with fluorescence and electron-paramagnetic resonance spectroscopy. Fluorescence spectroscopy revealed a superior correlation between observed swelling and solvation effects using a dansyl probe with JandaJel than with Merrifield resin. However, the internal viscosity of pre-swollen JandaJel is higher than Merrifield resin, as determined by EPR measurements. The combination of these two analytical methods provides insights into the physical differences observed between these two chemically similar resins and suggests caution should be used if using singular physical techniques to probe the microenvironment of polymeric matrixes.

Chemistry, Organic↗