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J A Fee

Publications and source records attributed to J A Fee.

At least 55 records · Page 3Linked to original sources
PubMed1991

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.

Amino Acid Sequence↗
PubMed1991

Regulation of sod genes in Escherichia coli: relevance to superoxide dismutase function.

This review is concerned with the effects of environmental perturbations on the expression of the two superoxide dismutase (SOD) genes in Escherichia coli (sodA, MnSOD; sodB, FeSOD). Early studies using SOD activity, showed that MnSOD levels respond to changes in oxygen tension, type of substrate, redox active compounds, iron concentration, the nature of the terminal oxidant, and the redox potential of the medium. FeSOD levels appeared nominally insensitive to these perturbations. More recent molecular genetic studies revealed that sodA expression is subject to regulation by three major regulatory systems: fur (ferric uptake regulation) and arcA arcB (aerobic respiratory control) mediate repression of sodA, while a relatively new system, soxR soxS (superoxide response), mediates activation of sodA expression. By contrast, sodB expression, which is much less studied at this time, appears to be positively activated in trans by fur. A rudimentary gene regulation model is presented which rationalizes past observations, is experimentally testable, and should serve as a guide to future research in this area.

Bacterial Outer Membrane Proteins↗
PubMed1991

Structure-function relationships in iron and manganese superoxide dismutases.

Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E. coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 A, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A. The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal. The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O2-, while other conserved residues maintain interchain packing interactions. Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O2- binds to the metal at a sixth coordination site without displacing the solvent ligand. Because crystals reduced with dithionite show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C. Bull and J.A. Fee (1985), Journal of the American Chemistry Society 107, 3295-3304) is unlikely to be one of the histidines which bind the metal ion. Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(III) and Fe(II) enzymes at neutral and high pH.

Amino Acid Sequence↗
PubMed1990

Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis.

In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.

Animals↗
PubMed1990

Novel effect of aromatic compounds on the iron-dependent expression of the Escherichia coli K12 manganese superoxide dismutase (sodA) gene.

In Escherichia coli, the superoxide dismutase genes (sodA and sodB) sense the availability of Fe through the action of the fur locus [E. C. Niederhoffer, C. M. Naranjo, K. L. Bradley, J. A. Fee (1990) Control of Escherichia coli superoxide dismutases (sodA and sodB) genes by the ferric uptake regulation (fur) locus, J. Bacteriol. 172, 1930-1938]. Previous work from other laboratories has shown that a variety of metal chelators and of redox-active aromatic compounds can dramatically induce expression of sodA. Here we show that non-redox-active, non-metal-chelating aromatic compounds also enhance expression of a chromosomal sodA gene fusion and that these effects are strongly modulated by the Fur phenotype (Fur +/-) and by the availability of iron in the culture medium. The compounds studied were ethidium bromide, hemin, 2,2'-bipyridine, 1,10-phenantroline, 4,7-phenantroline, rhodamine B1, rhodamine 6G, and, for comparison to previous studies, Paraquat.

Ethidium↗
PubMed1990

Plasmid-associated aggregation in Thermus thermophilus HB8.

Thermus thermophilus HB8, a moderate thermophile, exhibits visible aggregation when growing on a rich broth. Strain HB8 also contains two cryptic plasmids. We isolated cured strains from HB8 and observed that loss of the 47-MDa plasmid was correlated with loss of aggregation. An enrichment procedure was developed for aggregating cells and used to demonstrate that aggregation was restored upon transformation of a cured strain with plasmid DNA. The aggregation phenotype of transformed cells was variably stable; most did not retain either the plasmid or the phenotype for prolonged periods of growth. Hybridization experiments using a partial sequence from the 47-MDa plasmid suggested the presence of a repeated DNA sequence on this plasmid and on the chromosome. This is the first report of a phenotype associated with a plasmid from a Thermus strain.

Bacterial Adhesion↗
PubMed1990

Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus.

The ferric uptake regulation (fur) gene product participates in regulating expression of the manganese- and iron-containing superoxide dismutase genes of Escherichia coli. Examination of beta-galactosidase activity coded from a chromosomal phi(sodA'-'lacZ) fusion suggests that metallated Fur protein acts as a transcriptional repressor of sodA (manganese superoxide dismutase [MnSOD]). Gel retardation assays demonstrate high-affinity binding of pure, Mn2(+)-Fur protein to DNA fragments containing the sodA promoter. These data and the presence of an iron box sequence in its promoter strongly suggest that sodA is part of the iron uptake regulon. An sodB'-'lacZ fusion gene borne on either a low- or high-copy plasmid yielded approximately two- to threefold more beta-galactosidase activity in Fur+ compared with Fur- cells; the levels of activity depended only weakly on the growth phase and did not change during an extended stationary phase. Measurement of FeSOD activity in logarithmic growth phase and in overnight cultures of sodA and fur sodA backgrounds revealed that almost no FeSOD activity was expressed in Fur- strains, whereas wild-type levels were expressed in Fur+ cells. Fur+ and Fur- cells bearing the multicopy plasmid pHS1-4 (sodB+) expressed approximately sevenfold less FeSOD activity in the fur background, and staining of nondenaturing electrophoretic gels indicates that synthesis of FeSOD protein was greatly reduced in Fur- cells. Gel retardation assays show that Mn2(+)-Fur had a significantly higher affinity for the promoter fragment of sodB compared with that of random DNA sequences but significantly lower than for the promoter fragment of sodA. These observations suggest that the apparent positive regulation of sodB does not result exclusively from a direct interaction of holo (metallated) Fur itself with the sodB promoter. Nevertheless, the sodB gene also appears to be part of the iron uptake regulon but not in the classical manner of Fe-dependent repression.

Chromosomes, Bacterial↗
PubMed1989

Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters.

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.

Electron Transport Complex III↗
PubMed1989

Evidence for cytochrome oxidase subunit I and a cytochrome c--subunit II fused protein in the cytochrome 'c1aa3' of Thermus thermophilus. How old is cytochrome oxidase?

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme-binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II. With respect to the 16-S rRNA-derived phylogeny of procaryotes, the results show that the genetic information for an O2-reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.

Amino Acid Sequence↗
PubMed1989

An infrared study of the binding and photodissociation of carbon monoxide in cytochrome ba3 from Thermus thermophilus.

The C-O stretching frequencies of fully reduced carbonmonoxy cytochrome ba3, a newly discovered terminal oxidase of the bacterium Thermus thermophilus (Zimmermann, B.H., Nitsche, C.I., Fee, J.A., Rusnak, F., and Münck, E. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5779-5783), are studied by Fourier transform infrared spectroscopy. Multiple C-O frequencies are observed in the Fourier transform infrared spectra, indicating the presence of discrete interconverting conformers of the enzyme. Upon photolysis, the CO is shown to migrate exclusively to CuB+. Above 200 K, the CO returns to the heme a3 by a thermal process which follows simple first-order kinetics. The rate of the reaction was studied from 205 to 230 K and at 300 K, yielding the activation parameters delta H = 14.9 kcal/mol and delta S = -5 cal/mol/K. These are compared with previously determined activation parameters for CO recombination in mitochondrial cytochrome aa3 preparations (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 257, 1639-1650). We report the novel finding that CO remains bound to CuB+ at room temperature during continuous photolysis of cytochrome ba3, and we conjecture on the possible interference of copper-bound CO in "flow-flash" and "triple-trap" studies of cytochrome c oxidases.

Calorimetry↗
PubMed1988

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures.

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.

Amino Acid Sequence↗
PubMed1988

Properties of a copper-containing cytochrome ba3: a second terminal oxidase from the extreme thermophile Thermus thermophilus.

We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single approximately equal to 35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CuA in this protein. Quantitative EPR and Mössbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mössbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CuB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.

Copper↗
PubMed1987

Evidence for structural heterogeneities and a study of exchange coupling. Mössbauer studies of cytochrome c1aa3 from Thermus thermophilus.

We have studied samples of oxidized (as isolated) cytochrome c1aa3 from Thermus thermophilus in the pH range 5.7 to 9.3 with Mössbauer spectroscopy. In this pH range, the spectra of cytochromes c1 and a are independent of pH, whereas the spectra of cytochrome a3 are not. Most importantly, spectra taken in applied fields up to 6.0 T reveal the presence of multiple ferric forms of cytochrome a3. At any given pH value, at least two high-spin ferric cytochrome a3 species can be distinguished; in addition, most samples contain a low-spin ferric cytochrome a3 species (less than 20% of cytochrome a3). The Mössbauer spectra show clearly that all forms of cytochrome a3 are spin coupled (to CuB). We have analyzed the high field (H greater than or equal to 1.5 tesla) spectra of a sample at pH 6.5 in the framework of a model that considers isotropic exchange-coupling, JS1.S2, between a high-spin ferric (S1 = 5/2) cytochrome a3 and cupric CuB (S2 = 1/2). In strong applied fields, the spectra can be fitted with any value for J greater than or equal to 0.5 cm-1. In the strong coupling case (J/D1 approximately greater than 3), a zero field splitting parameter D1 approximately 2.5 cm-1 is required for cytochrome a3; this value is distinctly smaller than those observed for high-spin ferric hemes (4-20 cm-1). A model assuming weak coupling magnitude of J approximately 1 cm-1, yields D1 approximately 8 cm-1 and a parameter set for cytochrome a3 quite similar to that reported for metmyoglobin. A J-value of approximately 1 cm-1 does not demand the presence of a ligand bridging between cytochrome a3 and CuB.

Chemical Phenomena↗
PubMed1987

Comparison of the spin-lattice relaxation properties of the two classes of [2Fe-2S] clusters in proteins.

Two classes of [2Fe-2S] proteins have been defined according to the mean value gav of their g tensor components (Bertrand, P., Guigliarelli, B., Gayda, J.P., Beardwood, P. and Gibson, J.F. (1985) Biochim. Biophys. Acta 831, 261-266). To characterize their magnetic properties better, we have compared the spin-lattice relaxation behavior of typical proteins which belong to these two classes, namely Spirulina maxima and adrenal ferredoxin for the gav approximately 1.96 class, Thermus thermophilus Rieske protein and Pseudomonas putida benzene dioxygenase for the gav approximately 1.91 class. For all these proteins, the data support the existence of an efficient Orbach process in the highest temperature range, which allows the determination of the exchange coupling parameter, J. From the comparison of the J values obtained in each class, it is concluded that the structural factors which determine the value of the g tensor and the strength of the antiferromagnetic exchange interactions are different.

Animals↗
PubMed1987

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus.

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

Azotobacter↗
PubMed1986

Steady-state kinetic studies of superoxide dismutases. Saturative behavior of the copper- and zinc-containing protein.

The mechanism of the Cu-Zn-containing superoxide dismutase (SD) was studied using a stopped-flow spectrophotometric system capable of forming aqueous solutions of O2- having initial concentrations up to approximately 5 mM. By lowering the temperature to 5.5 degrees C, we were able to observe saturation of the enzyme. At 5.5 degrees C and pH 9.3, the Michaelis-Menten parameters extracted from the kinetic traces were turnover number (TN) approximately 1 X 10(6) s-1, Km approximately 3.5 X 10(-3) M. Under our conditions, the average rate at which O-2 binds to the active site, TN/Km is 0.26 X 10(9) M-1 s-1. TN was decreased in the presence of D2O, and a solvent isotope effect of TNH/TND approximately 3.6 was measured while TN/Km was essentially unaffected by D2O. TN was increased by the presence of the general acid, ND4+. These observations, by analogy to earlier work with Fe X SD from Escherichia coli (Bull, C., and Fee, J. A. (1985) J. Am. Chem. Soc. 107, 3295-3304), suggest that H2O serves to donate the protons required to form product H2O2. Values of Km and TN for the zinc-deficient enzyme were found to be approximately a factor of 2 less than those obtained for the holoenzyme under identical experimental conditions, whereas TN/Km was largely unchanged. The imidazolate bridge is thus not essential for catalytically competent extraction of a proton from the solvent.

Animals↗