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1.
J Med Chem ; 44(25): 4309-12, 2001 Dec 06.
Article in English | MEDLINE | ID: mdl-11728178

ABSTRACT

An approach to reduce the log P in a series of diacylglycerol (DAG)-lactones known for their high binding affinity for protein kinase C (PK-C) is presented. Branched alkyl groups with reduced lipophilicity were selected and combined with the replacement of the ester or lactone oxygens by NH or NOH groups. Compound 6a with an isosteric N-hydroxyl amide arm represents the most potent and least lipophilic DAG analogue known to date.


Subject(s)
4-Butyrolactone/chemistry , Diglycerides/chemical synthesis , Hydroxamic Acids/chemistry , Lactones/chemical synthesis , Protein Kinase C/chemistry , 4-Butyrolactone/analogs & derivatives , Diglycerides/chemistry , Drug Design , Isoenzymes/chemistry , Lactones/chemistry , Ligands , Models, Molecular , Molecular Conformation , Protein Binding , Structure-Activity Relationship
2.
Proc Natl Acad Sci U S A ; 98(24): 14126-31, 2001 Nov 20.
Article in English | MEDLINE | ID: mdl-11717466

ABSTRACT

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca(2+)-dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophin-deficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wild-type cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.


Subject(s)
Mitochondria, Heart/enzymology , Nitric Oxide Synthase/metabolism , Animals , Calcium/metabolism , Calcium Signaling , Dystrophin/genetics , Dystrophin/physiology , Electrochemistry , Electron Transport Complex IV/metabolism , Mice , Mice, Knockout , Nitric Oxide/biosynthesis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type I , Superoxides/metabolism
3.
J Med Chem ; 44(12): 1892-904, 2001 Jun 07.
Article in English | MEDLINE | ID: mdl-11384235

ABSTRACT

A small, focused combinatorial library encompassing all possible permutations of acyl branched alkyl chains-small and large, saturated and unsaturated-was generated from the active diacylglycerol enantiomer (S-DAG) to help identify the analogue with the highest binding affinity (lowest Ki) for protein kinase C (PK-C) combined with the minimum lipophilicity (log P). The selected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylglycerol (diC8) despite being 1.4 log units more hydrophilic. Compound 3B indeed represents the most potent, hydrophilic DAG ligand to date. With the help of a green fluorescent protein (GFP)-tagged PK-Calpha, 3B was able to translocate the full length protein to the membrane with an optimal dose of 100 microM in CHO-K1 cells, while diC8 failed to achieve translocation even at doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of PK-Cdelta clearly showed the existence of a preferred binding orientation. In addition, molecular dynamic simulations suggest that binding discrimination could result from a favorable van der Waals (VDW) interaction between the large, branched sn-1 acyl group of 3B and the aromatic rings of Trp252 (PK-Cdelta) or Tyr252 (PK-Calpha). The DAG analogue of 3B in which the acyl groups are reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively discriminate between alternative orientations of the acyl chains.


Subject(s)
Diglycerides/chemistry , Diglycerides/pharmacology , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Animals , Binding Sites , Binding, Competitive , CHO Cells , Cricetinae , Databases as Topic , Diglycerides/chemical synthesis , Enzyme Activation , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Conformation , Phorbol 12,13-Dibutyrate/pharmacokinetics , Protein Conformation , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Transfection , Tryptophan , Tyrosine , Zinc Fingers
4.
Nitric Oxide ; 5(2): 128-36, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11292362

ABSTRACT

In 32D cl 3 hematopoietic progenitor cells, the overexpression of manganese superoxide dismutase (MnSOD, SOD2), the enzyme normally found in mitochondria, protects against the damaging effects of ionizing radiation. In the presence of a nitric oxide donor, which exacerbates the damage, inhibition of mitochondrial function can be demonstrated to be associated with respiratory complexes I (NADH dehydrogenase) and III (cytochrome c reductase), but not II (succinate dehydrogenase), IV (cytochrome c oxidase), or V (ATP synthase). The same pattern of inhibition is observed in the case of isolated bovine heart mitochondria exposed to ionizing radiation and the nitric oxide donor. The addition of authentic peroxynitrite (ONO2(-)) to isolated mitochondria also results in damage to complexes I and III (but not II, IV, and V), as shown by assays of electron-transfer activities and electron paramagnetic resonance (EPR) spectroscopic measurements, suggesting ONO2(-) to be responsible for most of the observed radiation damage in both the cultured cell lines and isolated mitochondria. It is argued that, in general, production of ONO2(-) is an important contributor to radiation damage in biological systems and the implications of these findings in relation to possible mechanisms of oxidant-linked apoptosis are briefly considered.


Subject(s)
Mitochondria/drug effects , Mitochondria/radiation effects , NADH Dehydrogenase/metabolism , Nitric Oxide/pharmacology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Cattle , Cell Line , Electron Spin Resonance Spectroscopy , Electron Transport/drug effects , Half-Life , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/radiation effects , Intracellular Membranes/drug effects , Intracellular Membranes/pathology , Mitochondria/enzymology , Mitochondria/pathology , Nitrates/metabolism , Nitrates/pharmacology , Nitric Oxide/metabolism , Oxidants/metabolism , Oxidants/pharmacology , Oxidative Stress/drug effects , Rabbits , Radiation, Ionizing , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxides/metabolism
5.
J Nutr ; 130(5S Suppl): 1467S-70S, 2000 05.
Article in English | MEDLINE | ID: mdl-10801961

ABSTRACT

Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N, N,N',N'-tetrakis(2. pyridylmethyl)ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic Ca(2+) via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall.


Subject(s)
Antioxidants/pharmacology , Endothelium, Vascular/physiology , Homeostasis/physiology , Metallothionein/physiology , Nitric Oxide/pharmacology , S-Nitrosothiols , Zinc/physiology , Animals , Cells, Cultured , Chelating Agents/metabolism , Chelating Agents/pharmacology , Cysteine/analogs & derivatives , Cysteine/pharmacology , Drug Interactions , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Ethylenediamines/metabolism , Ethylenediamines/pharmacology , Fluorescent Dyes/metabolism , Fluorescent Dyes/pharmacology , Homeostasis/drug effects , Mice , Mice, Knockout , Nitroso Compounds/pharmacology , Oxidation-Reduction/drug effects , Pulmonary Artery , Quinolones/metabolism , Quinolones/pharmacology , Sheep , Tosyl Compounds/metabolism , Tosyl Compounds/pharmacology , Zinc/pharmacology
6.
Proc Natl Acad Sci U S A ; 97(1): 477-82, 2000 Jan 04.
Article in English | MEDLINE | ID: mdl-10618443

ABSTRACT

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(-/-)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.


Subject(s)
Luminescent Proteins/genetics , Metallothionein/metabolism , Nitric Oxide/metabolism , Animals , Arginine/pharmacology , Calcium/metabolism , Endothelium, Vascular/metabolism , Glutathione/analogs & derivatives , Glutathione/metabolism , Green Fluorescent Proteins , Image Processing, Computer-Assisted , Kinetics , Male , Mesenteric Arteries , Metallothionein/genetics , Mice , Mice, Inbred Strains , Mice, Knockout , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitroso Compounds/metabolism , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S-Nitrosoglutathione , Signal Transduction , Spectrometry, Fluorescence
7.
J Biol Chem ; 274(50): 35763-7, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10585458

ABSTRACT

Fully and partially reduced forms of isolated bovine cytochrome c oxidase undergo a two-electron oxidation-reduction process with added peroxynitrite, leading to catalytic oxidation of ferrocytochrome c to ferricytochrome c. The other major reaction product is nitrite ion, 86% of the added peroxynitrite being measurably converted to this species. The reaction is inhibited in the presence of cyanide, implicating the heme a(3)-Cu(B) binuclear pair as the active site. Moreover, provided peroxynitrite is not added to excess, the reductase activity of the enzyme toward this oxidant efficiently protects other protein and detergent molecules in vitro from nitration of tyrosine residues and oxidative damage. If the enzyme is exposed to approximately 10(2)-fold excesses of peroxynitrite, then significant irreversible loss of electron transfer activity results, and the heme a(3)-Cu(B) binuclear pair no longer undergo a characteristic carbon monoxide-driven reduction. The accompanying rather small changes in the observed electronic absorption spectrum are suggestive of a modification in the vicinity of one or both hemes but probably not to the cofactors themselves.


Subject(s)
Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Oxidoreductases/metabolism , Animals , Carbon Monoxide/metabolism , Cattle , Copper/metabolism , Electron Transport , Heme/metabolism , Kinetics , Mitochondria, Heart/enzymology , Nitrates/metabolism , Oxidants/metabolism , Oxidation-Reduction , Oxidoreductases/chemistry , Spectrophotometry
8.
J Biol Chem ; 274(9): 5499-507, 1999 Feb 26.
Article in English | MEDLINE | ID: mdl-10026163

ABSTRACT

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.


Subject(s)
Carbon Monoxide/chemistry , Copper/chemistry , Cysteine/chemistry , Heme/chemistry , Methemoglobin/chemistry , Adult , Electron Spin Resonance Spectroscopy , Electron Transport , Humans , Kinetics , Oxidation-Reduction
9.
Antioxid Redox Signal ; 1(3): 349-64, 1999.
Article in English | MEDLINE | ID: mdl-11229446

ABSTRACT

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The roles of MT in copper (Cu) binding and release and modulation of redox cycling are unresolved. We hypothesized that Cu-binding to MT renders Cu redox inactive, but that oxidation of free thiols critical for metal binding can reduce MT/Cu interactions and potentiate Cu redox cycling. Overexpression of MT in cells by cadmium pretreatment or ectopic overexpression by gene transfer confers protection from Cu-dependent lipid oxidation and cytotoxicity. Using a chemically defined model system (Cu/ascorbate/H2O2) to study Cu/MT interactions, we observed that MT inhibited Cu-dependent oxidation of luminol. In the absence of H2O2, MT blocked Cu-dependent ascorbyl radical production with a stoichiometry corresponding to Cu/MT ratios < or = 12. In the presence of H2O2, Cu-dependent hydroxyl radical formation was inhibited only up to Cu/MT ratios < or = 6. Using low-temperature EPR of free Cu2+ to assess Cu/MT physical interactions, we observed that the maximal amount of Cu1+ bound to MT corresponded to 12 molar equivalents of Cu/MT with Cu and ascorbate alone and was reduced in the presence of H2O2. 2,2'-Dithiodipyridine titration of MT SH-groups revealed a 50% decrease after H2O2, which could be regenerated by dihydrolipoic acid (DHLA). DHLA regeneration of thiols in MT was accompanied by restoration of MT's ability to inhibit Cu-dependent oxidation of ascorbate. Thus, optimum ability of MT to inhibit Cu-redox cycling directly correlates with its ability to bind Cu. Some of this Cu, however, appears releasable following oxidation of the thiolate metal-binding clusters. We speculate that redox-dependent release of Cu from MT serves both as a mechanism for physiological delivery of Cu to specific target proteins, as well as potentiation of cellular damage during oxidative stress.


Subject(s)
Antioxidants/metabolism , Copper/metabolism , Copper/pharmacology , Metallothionein/metabolism , Oxidants/metabolism , Thioctic Acid/analogs & derivatives , Animals , Antioxidants/pharmacology , Cadmium/pharmacology , Cell Survival , Copper/antagonists & inhibitors , Copper/toxicity , Electron Spin Resonance Spectroscopy , Endothelium, Vascular/drug effects , HL-60 Cells , Humans , Hydrogen Peroxide/pharmacology , Lipid Peroxidation/drug effects , Liver , Luminol/metabolism , Metallothionein/genetics , Metallothionein/pharmacology , Oxidants/pharmacology , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Protein Binding/drug effects , Rabbits , Sulfhydryl Compounds/metabolism , Thioctic Acid/pharmacology , Transfection
10.
Comp Biochem Physiol B Biochem Mol Biol ; 114(4): 345-52, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8840511

ABSTRACT

Contrary to previous reports, the functional and spectral properties of "monomeric" shark cytochrome c oxidases are not entirely similar to those of the "dimeric" beef enzyme. Most significantly, unlike the behavior of beef oxidase, the fully oxidized shark enzyme is not reducible by carbon monoxide. Also, preparations of the shark enzyme, isolated at pH 7.8-8.0, lead to more than 60% of the sample always being obtained in a resting form, whereas similarly prepared beef oxidase is very often obtained, both by ourselves and others, exclusively in the pulsed form. Although the electronic absorption, magnetic circular dichroism and electron paramagnetic resonance (EPR) spectra of cytochrome c oxidase obtained from several shark species are similar to those of the beef enzyme, there are some significant differences. In particular, the Soret maximum is at 422 nm in the case of the fully oxidized resting shark oxidases at physiological pH and not 418 nm as commonly found for the beef enzyme. Moreover, the resting shark oxidases do not necessarily exhibit a "g = 12" signal in their EPR spectra. The turnover numbers of recent preparations of the shark enzyme are higher than previously reported and, interestingly, do not differ within experimental uncertainty from those documented for several beef isoenzymes assayed under comparable conditions.


Subject(s)
Carbon Monoxide/metabolism , Electron Transport Complex IV/metabolism , Animals , Circular Dichroism , Cyanides/metabolism , Electron Spin Resonance Spectroscopy , Electron Transport Complex IV/chemistry , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Muscle, Skeletal/enzymology , Myocardium/enzymology , Protein Conformation , Sharks
11.
Biochem J ; 305 ( Pt 3): 871-8, 1995 Feb 01.
Article in English | MEDLINE | ID: mdl-7848288

ABSTRACT

A protocol for obtaining high-quality saturation-magnetization data from metalloprotein samples, employing a superconducting quantum interference device (SQUID) magnetometer, has previously been reported [E. P. Day, T. A. Kent, P. A. Lindahl, E. Münck, W. H. Orme-Johnson, H. Roder and A. Roy (1987) Biophys. J. 52, 837-853 and E. P. Day (1993) Methods Enzymol. 227, 437-463]. Following studies of several dozen different metalloprotein derivatives, the methodology has been further refined, particularly in the area of sample preparation. The details of the sample-handling procedures now in use are described, and moreover, the critical issue of verifying that contamination by paramagnetic impurities remains insignificant is considered. Importantly, it is shown that an independent determination of the quantity of paramagnetic sample present in the magnetometer is undesirable. Much more reliable parameters concerning the ground-state magnetic properties of the system under study are obtained if enough saturation-magnetization data are collected to enable the spin concentration to be determined during the subsequent fitting procedure. As proof of the validity of this method, the results of magnetization studies on ferricytochrome c, ferrocytochrome c and the benzohydroxamic acid adduct of horseradish peroxidase are presented. The ability of saturation-magnetization measurements to routinely determine spin concentration to within +/- 4% of accepted values is firmly established. In addition, a saturation-magnetization study has been performed on resting and fully reduced derivatives of cytochrome c oxidase. These results provide an illustration of the usefulness of the technique in probing some systems which have proved difficult to study by other methods. The increased difficulties inherent in obtaining meaningful data from these cytochrome c oxidase and other integer spin systems are delineated.


Subject(s)
Electron Spin Resonance Spectroscopy , Electron Transport Complex IV/chemistry , Magnetics , Metalloproteins/chemistry , Animals , Cattle , Copper/chemistry , Cytochrome c Group/chemistry , Dithionite , Edetic Acid , Heme/chemistry , Horseradish Peroxidase/chemistry , Hydroxamic Acids/chemistry
12.
Biophys J ; 67(2): 713-9, 1994 Aug.
Article in English | MEDLINE | ID: mdl-7948684

ABSTRACT

Computer simulations of dipalmitoylphosphatidylcholine (DPPC) have been performed using Langevin dynamics and a Marcelja-type mean field. This work has focused on the dynamics of the choline head group to parameterize the empirical constraints against phosphorus-carbon dipolar couplings (Dp-c) as measured by nuclear magnetic resonance (13C-NMR). The results show good agreement with experimental values at constraints equivalent to the choline tilt observed in joint refinement of x-ray diffraction and neutron diffraction scatterings. Quadrupolar splittings for the alpha and beta positions are also calculated and compared with 2H-NMR experiments. The model predicts torsional transition rates around the alpha-beta bonds and for the two C-O-P-O torsions. It also predicts T1 relaxation times for the alpha and beta carbons.


Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Algorithms , Choline , Computer Simulation , Magnetic Resonance Spectroscopy , Molecular Conformation
13.
Biophys J ; 65(3): 1084-92, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8241389

ABSTRACT

Computer simulations of three unsaturated phospholipids in a membrane environment have been carried out using Langevin dynamics and a mean-field based on the Marcelja model. The applicability of the mean-field to model unsaturated lipids was judged by comparison to available experimental NMR data. The results show that the mean-field methodology and the parameters developed for saturated lipids are applicable in simulations of unsaturated molecules, indicating that these simulations have good predictive capabilities. Single molecule simulations, each 100 ns in length, of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-elaidoyl-sn-glycero-3-phosphocholine (PEPC), and 1-palmitoyl-2-isolinoleoyl-sn-glycero-3-phosphocholine (PiLPC) reveal similarities between PEPC and DPPC. The presence of the trans double bond in PEPC has a minimum impact on the structural and dynamic properties of the molecule, which is probably the reason that isolated trans double bonds are rare in biological lipids. POPC exhibits different behavior, especially in the calculated average interchain distances, because of the cis double bond. The position of the two double bonds in PiLPC imparts special properties to the molecule.


Subject(s)
Membranes, Artificial , Phospholipids/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Algorithms , Biophysical Phenomena , Biophysics , Computer Simulation , Magnetic Resonance Spectroscopy , Models, Chemical , Phosphatidylcholines/chemistry , Stereoisomerism , Thermodynamics
14.
Biochemistry ; 29(48): 10847-54, 1990 Dec 04.
Article in English | MEDLINE | ID: mdl-2271684

ABSTRACT

X-ray crystallographic studies of the intradiol cleaving protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have shown that the enzyme has a trigonal bipyramidal ferric active site with two histidines, two tyrosines, and a solvent molecule as ligands [Ohlendorf, D.H., Lipscomb, J.D., & Weber, P.C. (1988) Nature 336, 403-405]. Fe K-edge EXAFS studies of the spectroscopically similar protocatechuate 3,4-dioxygenase from Brevibacterium fuscum are consistent with a pentacoordinate geometry of the iron active site with 3 O/N ligands at 1.90 A and 2 O/N ligands at 2.08 A. The 2.08-A bonds are assigned to the two histidines, while the 1.90-A bonds are associated with the two tyrosines and the coordinated solvent. The short Fe-O distance for the solvent suggests that it coordinates as hydroxide rather than water. When the inhibitor terephthalate is bound to the enzyme, the XANES data indicate that the ferric site becomes 6-coordinate and the EXAFS data show a beat pattern which can only be simulated with an additional Fe-O/N interaction at 2.46 A. Together, the data suggest that the oxygens of the carboxylate group in terephthalate displace the hydroxide and chelate to the ferric site but in an asymmetric fashion. In contrast, protocatechuate 3,4-dioxygenase remains 5-coordinate upon the addition of the slow substrate homoprotocatechuic acid (HPCA). Previous EPR data have indicated that HPCA forms an iron chelate via the two hydroxyl functions.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Brevibacterium/enzymology , Iron/chemistry , Protocatechuate-3,4-Dioxygenase/chemistry , 3,4-Dihydroxyphenylacetic Acid/metabolism , Binding Sites , Chemical Phenomena , Chemistry, Physical , Fourier Analysis , Histidine/chemistry , Molecular Structure , Nitrogen/chemistry , Oxygen/chemistry , Phthalic Acids/metabolism , Protocatechuate-3,4-Dioxygenase/metabolism , X-Ray Diffraction
15.
Biochemistry ; 29(40): 9365-9, 1990 Oct 09.
Article in English | MEDLINE | ID: mdl-2174257

ABSTRACT

Replacement of Phe-82 in yeast iso-1-cytochrome c with Tyr, Leu, Ile, Ser, Ala, and Gly produces a gradation of effects on (1) the reduction potential of the protein, (2) the rate of reaction with Fe(EDTA)2-, and (3) the CD spectra of the ferricytochromes in the Soret region under conditions where contributions from the alkaline forms of these proteins are absent. The reduction potential of cytochrome c is lowered by as little as 10 mV (Tyr-82) or by as much as 43 mV (Gly-82; pH 6.0) as the result of these substitutions. The second-order rate constants for reduction of these cytochromes range from a low of 6.20 (2) x 10(4) for the Tyr-82 variant to a high of 14.8 x 10(4) M-1 s-1 for the Ser-82 variant [pH 6.0, 25 degrees C, mu = 0.1 M (sodium phosphate)]. Analysis of these rates by use of relative Marcus theory produces values of k11corr that range from 10.9 M-1 s-1 for the wild-type protein to 190 M-1 s-1 for the Gly-82 mutant [25 degrees C, mu = 0.1 M, pH 6.0 (sodium phosphate)]. Reinvestigation of the effect of substituting Phe-82 by a Tyr residue on the CD spectrum of the protein now reveals little alteration of the intense, negative Cotton effect in the Soret CD spectrum of ferricytochrome c. On the other hand, substitution of nonaromatic residues of various sizes at this position results in loss of this spectroscopic feature, consistent with previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cytochrome c Group/metabolism , Cytochromes c , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Circular Dichroism , Cytochrome c Group/chemistry , Cytochrome c Group/genetics , Edetic Acid , Electrochemistry , Kinetics , Mutation , Oxidation-Reduction , Protein Conformation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
16.
Biochemistry ; 28(8): 3152-6, 1989 Apr 18.
Article in English | MEDLINE | ID: mdl-2545249

ABSTRACT

The possible influence of residue Phe-82 in the cytochrome c alkaline isomerization has been evaluated by spectrophotometric pH titrations of a family of mutant yeast iso-1-cytochromes c in which the identity of the residue at this position has been varied. The pKa for the exchange of the Met-80 heme iron ligand was determined from pH titrations in which the S----Fe charge-transfer band (695 nm) was monitored and was found to be 8.5 for the wild type, 7.7 for Ser-82, 7.7 for Gly-82, 7.2 for Leu-82, and 7.2 for Ile-82. pH-jump experiments [Davis et al. (1974) J. Biol. Chem. 249, 2624] established that substitutions at position 82 affect the alkaline isomerization by lowering the pKa of the titrating group by as much as 1.4 pK units; for the Ser-82 and Gly-82 variants, there is also a small effect on the Keq for the ligand exchange equilibrium. On the basis of these findings, we conclude that one critical role for Phe-82 in the wild-type protein is stabilization of the native heme binding environment.


Subject(s)
Cytochrome c Group/metabolism , Cytochrome c Group/genetics , Heme/metabolism , Hydrogen-Ion Concentration , Isomerism , Kinetics , Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
17.
Biochemistry ; 26(26): 8709-17, 1987 Dec 29.
Article in English | MEDLINE | ID: mdl-2831950

ABSTRACT

We have examined the effects on redox kinetics of changing the reduction potential of the mu-oxo-bridged binuclear iron center in octameric hemerythrin (Hr) from Phascolopsis gouldii. The opportunity to examine such effects is provided by the availability of mu-sulfidomethemerythrin (mu-S2-metHr), whose [Fe(III),Fe(III)]met----[Fe(II),Fe(III)]semi-met reduction potential is approximately 200 mV higher than that of methemerythrin (metHr). We have used, as redox partners to Hr, a set of metal complexes and the heme proteins deoxymyoglobin (Mb) and cytochrome b5. The latter protein from P. gouldii is a presumed physiological redox partner of Hr. Similar kinetics at pH 8 in the presence or absence of the allosteric effector perchlorate suggest reduction of the iron atom closer to the outer surface of each subunit in the Hr octamer during the met----semi-met transformation. For all reducing agents, the experimentally determined ratio of second-order rate constants for reductions of mu-S2-metHr and metHr, k12(mu-S2-met)/k12(met), is close to the value of 40 predicted by the simple Marcus relation for "outer-sphere" electron transfer. For oxidations of (semi-met)RHr and mu-S2-semi-metHr, the predicted value of 40 for k12[(semi-met)R]/k12(mu-S2-semi-met) is closely approximated when Fe(CN)6(3-) is used as oxidant. The ionic strength dependence of the second-order rate constant suggests electrostatic interactions of opposite charges during reduction of metHr by P. gouldii cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cytochrome b Group/metabolism , Hemerythrin/metabolism , Metalloproteins/metabolism , Animals , Calorimetry , Cattle , Cytochromes b5 , Electron Spin Resonance Spectroscopy , Hemerythrin/analogs & derivatives , Kinetics , Liver/metabolism , Macromolecular Substances , Models, Molecular , Nematoda/metabolism , Oxidation-Reduction , Protein Conformation
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