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We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered complete protection from Fe2+/ascorbate-induced inactivation. The substrates PEP and GTP, but not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic studies indicate that the inactive enzyme no longer binds Mn2+ but still had high affinity for GTP indicating that the inactivation process was specific for the metal site. A decrease in cysteine content was observed over time following PEPCK treatment with Fe2+ and ascorbate. The apparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min-1) is similar to the apparent first-order rate constant for inactivation (0.067 +/- 0.005 min-1). Amino acid composition analysis revealed that cysteic acid was generated upon Fe2+/ascorbate addition to PEPCK. Native chicken liver PEPCK has an Mr of 67 kDa. SDS-PAGE of the inactivated enzyme showed the presence of two new bands at 31.7 and 35.3 kDa indicating that PEPCK was specifically cleaved at a single site. The rate of cleavage was slower than the rate of inactivation and fully inactivated enzyme was only 50% cleaved. The Fe2+/ascorbate-catalyzed inactivation was not solely due to protein cleavage. The protein fragments generated by cleavage were separated by C4 reverse phase HPLC. The cleavage exposed a new N-terminus which was identified to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragments indicated that the site of cleavage was between Asp296 and Ile297. These results indicate that Asp296 is involved in metal chelation. This agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) Biochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are involved in metal binding.

Tyramine β-monooxygenase (TβM) belongs to a small class of eukaryotic copper-, ascorbate-, and O2-dependent enzymes that includes dopamine β-monooxygenase (DβM) and peptidylglycine α-hydroxylating monooxygenase (PHM) (1-3). TβM is the insect homologue of DβM and plays a neuroregulatory role in insects by catalyzing the β-hydroxylation of tyramine to yield octopamine (eq. 1) (4-6). (1) Studies indicate that TβM expression occurs in octopaminergic neurons (5,6). A significant amount of spectroscopic and kinetic data collected for these enzymes has demonstrated a high conservation of mechanism (7-14). Structural information about TβM is inferred from primary amino acid sequence homology and the X-ray structures solved for PHM (3,15-17). TβM is assumed to use two solvent-exposed, non-coupled mononuclear copper centers, denoted CuM and CuH and separated by ~ 11 A (Figure 1), to incorporate an oxygen atom into the phenethylamine side chain of tyramine (3,14), eq. 1. O2 activation and substrate hydroxylation occur exclusively at the CuM domain, which is coordinated by two histidines and a Met residue (17,18). CuH, which is ligated by three histidine residues, serves as an electron reservoir to supply the second electron required for insertion of oxygen into the C–H bond by long-range intramolecular electron transfer (7). The combination of structural, kinetic, biochemical, and theoretical evidence supports the formation of a CuM-superoxo intermediate as the active oxygen species responsible for C–H activation at the CuM site (1,17,19-22); by contrast, the precise timing and pathway for electron transfer from CuH to CuM remain under active debate (19,20,22). Figure 1 (Recreated from PDB: 1PHM) (15). The ligands at CuH (His107, His108 and His172) and to CuM (His242, His244, Met314) of PHM. Met471 in TβM corresponds to Met314 in PHM. The Met residue at the CuM domain appears to be a critical component for catalysis by these enzymes, but the exact function of this residue is not known. The ability of the CuM-Met ligand to move into the coordination sphere upon reduction of the copper domains is supported by spectroscopic and crystallographic analyses (2,7,9-11,15,23). Extended X-ray absorption fine structure (EXAFS) for the Cu(II) state of PHM, DβM and TβM indicates an average of 2.5 nitrogen (His) and 1.5 oxygen or nitrogen ligands per copper at 1.97 A (2,7,10,11,23) and a weak sulfur ligand detected at 2.71 A, in agreement with the PHM X-ray crystal structure (15). Enzyme reduction leads to a shorter Met to copper bond distance: 2.27 A in PHM, 2.25 A in DβM, and 2.24 A in TβM (2,10,11,23). Importantly, despite the active site solvent exposure of these enzymes and the requirement for cycling between Cu(I) and Cu(II) during each catalytic cycle, the consumption of O2 and the generation of hydroxylated product are tightly coupled. Neither the use of a very slow substrate with DβM (19) or site-specific mutation in PHM (His172Ala) (20), both of which diminished kcat by nearly three orders of magnitude, has any impact on the 1:1 ratio for O2 uptake and product formation. The aggregate data implicate a dynamic role for the Met ligand to CuM that regulates the equilibrium for generating a copper-superoxo species and prevents the diffusional loss of activated oxygen species during turnover (1,19,22). Site-directed mutagenesis has been used extensively to understand the significance of the Met ligand to CuM. Studies with PHM in which Met314 was substituted with Ile, His, Asp, and Cys, reported that the hydroxylation of glycine-extended peptides was undetectable in spent media (2,24), whereas a low activity has been reported for Met314His (25). The X-ray structure of the Met314Ile PHM variant was found to lead to a significant disorder at the CuH domain, implicating communication between the ligands at distal metal sites (26). Recently, the analogous Met ligand in TβM was mutated to a Cys, His, and Asp, and only the Met471Cys retained measureable activity. Electron paramagnetic resonance (EPR) characterization of the Cu(II) forms of WT and Met471 variants have shown identical properties. Interestingly, Met471Cys TβM was unable to fully convert substrate to product under any conditions examined. This result led to the proposal of a secondary inactivation pathway during turnover (27). XAS/EXAFS experiments were carried out on the series of Met471 variants, to examine how changes in structure relate to the observed altered reactivity, and these showed no binding of His and Asp to Cu(I)M and only a very weak/small interaction between Cys and the metal site (23). Herein, we demonstrate that the replacement of Met471 by Cys generates an enzyme form that undergoes directed oxidation at position 471. This is attributed to the poor coordination between Cys471 and Cu(I)M, and not to a substrate-dependent inactivation as previously suggested (27).

Isopenicillin N synthase (IPNS) catalyzes double ring closure of the tripeptide (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of penicillin-type antibiotics. Our previous spectroscopic study using IPNS from Cephalosporium acremonium expressed in Escherichia coli [Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Munck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681] indicated that a thiolate enters the coordination of the essential active site Fe2+ when ACV binds to IPNS. The presence of an Fe-S bond in the IPNS.ACV complex is confirmed by EXAFS data presented in the preceding paper [Scott, R. A., Wang, S., Eidsness, M. K., Kriauciunas, A., Frolik, C. A. & Chen, V. J. (1992) Biochemistry (preceding paper in this issue)]. However, these studies leave unclear whether the coordinating thiolate derives from ACV or an endogenous cysteine. Here, we examine the spectroscopic properties of three genetically engineered variants of IPNS in which the only two endogenous cysteines are individually and collectively replaced by serine. The EPR, Mossbauer, and optical spectra of the mutant enzymes and their complexes with ACV, NO, or both ACV and NO are found to be essentially the same as those of wild-type IPNS, showing that the endogenous cysteines are not Fe2+ ligands in any of these complexes. Spectral quantitations show that the double Cys----Ser mutation decreases the affinity of the enzyme for ACV by about 6-fold, suggesting that the endogenous cysteines influence the structure of the substrate binding pocket remote from the iron. Thiolate complexation of the Fe2+ is also examined using ACV analogues. All ACV analogues examined in which the cysteinyl thiol moiety is unaltered are found to bind to the IPNS.NO complex to give optical and EPR spectra very similar to those of the ACV complex. In contrast, analogues in which the cysteinyl moiety of ACV is replaced with serine or cysteic acid fail to elicit the characteristic EPR and optical features despite the fact that they are bound with reasonable affinity to the enzyme. These results demonstrate that the thiolate of ACV coordinates the Fe2+. The EPR spectra of both the IPNS.NO and IPNS.ACV.NO complexes are broadened for samples prepared in 17O-enriched water, showing that water (or hydroxide) is also an iron ligand in each case. Thus, the Fe2+ coordination of the IPNS.ACV.NO complex accommodates at least three exogenous ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.

Chlorobium limicola, strain Tassajara, cytochrome c-551 is a soluble dimeric protein containing identical subunits of about 30 kDa. The amino acid sequence was determined by a combination of automated Edman degradation and mass analysis. There are 258 residues with a single heme binding site located at cysteine positions 172 and 175. In addition, there is a disulfide bridge between Cys78 and Cys109, and a free cysteine at position 219 which was found to occur as cysteic acid. The only homologue of soluble cytochrome c-551 is the soxA protein which is part of the thiosulfate utilization operon of Paracoccus denitrificans. They are 32% identical with three small gaps. This is consistent with the observation that cytochrome c-551 is the electron acceptor for a thiosulfate-oxidizing enzyme. On the basis of the redox potential of 135 mV, the sixth heme ligand should be a methionine. Among the seven methionine residues that are present in c-551, only one is conserved, two residues ahead of the heme-binding site. The far-UV circular dichroism spectrum indicates 40% alpha helix and 25% beta secondary structure. No other known cytochrome c has such a mixed structure; they are either all helical or all beta. Thus, Chlorobium soluble cytochrome c-551 and soxA are likely to be representative of a new class of c-type cytochromes.

Several methods were used to analyze for tryptophan in the acetylcholine (ACh) receptors purified from the electric organs of the electric rays, Torpedo californica and Torpedo marmorata. The best value of tryptophan was 2.4 mol %. When excited at 290 nm, both receptors fluoresced with a maximum at 336, but there was no change in the fluorescence emission spectra upon binding of carbamylcholine, d-tubocurarine, ACh, or decamethonium. The free SH content of the Torpedo receptors varied in different preparations, and was highest in that purified from fresh T. californica using deaerated solutions and dialysis under nitrogen, and lowest in that prepared from the aged lyophilized membranes of T. marmorata. The maximum free SH content was 20 nmol/mg of protein or 0.22 mol %, equal to at most 18% of the total cysteic acid residues. Reaction of either 33% or of all the SH residues with p-chloromercuribenzoate reduced maximum ACh binding to the pure receptor prepared from fresh T. californica by only 23%.

Kinetic studies have been made with glutathione-insulin transhydrogenase, an enzyme which degrades insulin by promoting cleavage of its disulfide bonds via sulfhydryl-disulfide interchange. The degradation of 125I-labeled insulin by enzyme purified from beef pancreas was studied with various thiol-containing compounds as cosubstrates. The apparent Km for insulin was found to be a function of the type and concentration of thiol; values obtained were in the range from 1 to 40 muM. Lineweaver-Burk plots for insulin as varied substrate were linear, whereas those for the thiol substrates were nonlinears: the plots for low molecular weight monothiols (GSH and mercaptoethanol) were parabolic; those for low molecular weight dithiols (dithiothreitol, dihydrolipoic acid, and 2,3-dimercaptopropanol) were apparently linear modified by substrate inhibition; and the plots for protein polythiols (reduced insulin A and B chains and reduced ribonuclease) were parabolic with superposed substrate inhibition. The nonparallel nature of the reciprocal plots for all substrates shows that the enzyme does not follow a ping-pong mechanism. Product inhibition studies were performed with GSH as thiol substrate. Oxidized glutathione was found to be a linear competitive inhibitor vs. both GSH and insulin. The S-sulfonated derivative of insulin A chain was also linearly competitive vs. both substrates. Inhibition by S-sulfonated B chain was competitive vs. insulin; the data eliminated the possibility that this derivative was uncompetitive vs. GSH. Experiments with the cysteic acid derivatives of insulin A and B chains similarly excluded the possibility that these were uncompetitive vs. either substrate. These inhibition studies indicate that the enzyme probably follows a randdom mechanism.