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PubMed1984

Patterns of product inhibition for bacterial nitrite reductase.

Product inhibition has been examined in the turnover kinetics of cytochrome cd1 from Pseudomonas aeruginosa (ATCC 19429) and from Paracoccus denitrificans1 (ATCC 13456). A common characteristic was a decrease in rate during the time course of assays that was not due to substrate depletion or irreversible inactivation. The product of nitrite reduction, nitric oxide (NO), acted as a product inhibitor in anaerobic assays with an apparent Ki of 0.2 microM, but only if the enzyme was first preincubated with NO for 15 min. The enzyme was inhibited by the oxidized form of electron donors and this could account for the decrease in rate during an assay. For the donors hydroquinone, ascorbate, TMPD, and azurin, measured values of the inhibition constant were at least ten fold lower than measured Km's. Cytochromes c as donors demonstrated a complex pattern of product inhibition by the ferric form. Although numerical values of Ki in these cases were not obtained, trends indicated that apparent values would be less than Km.

Aerobiosis↗
PubMed1981

Structural features and the reaction mechanism of cytochrome oxidase: iron and copper X-ray absorption fine structure.

X-ray edge absorption of copper and extended fine structure studies of both copper and iron centers have been made of cytochrome oxidase from beef heart, Paracoccus dentrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme). The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the x-ray beam was controlled by low temperature (-140 degrees C) and was continuously monitored by simultaneous optical spectroscopy and by electron paramagnetic resonance (EPR) monitoring every 30 min of x-ray exposure. The structure of the active site, a cytochrome a3-copper pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a sulphur bridge with three ligands 2.60 +/- 0.03 A from Fea3 and 2.18 +/- 0.03 A from Cua3. The distance between Fea3 and Cua3 is 3.75 +/- 0.05 A, making the sulphur bond angle 103 degrees reasonable for sp3 sulphur bonding. The Fea3 first shell has four typical heme nitrogens (2.01 +/- 0.03 A) with a proximal nitrogen at 2.14 +/- 0.03 A. The sixth ligand is the bridging sulphur. The Cua3 first shell is identical to oxidized stellacyanin containing two nitrogens and a bridging sulphur. Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cua3 first shell and contains the sulphur that forms the bridge in fully oxidized and mixed valence formate states. The Fea3 first shell is identical to oxyhemoglobin but has CO instead of O2. The other redox centers, Fea and the other "EPR detectable" Cu are not observed in higher shells of Fea3. Fea has six equidistant nitrogens and Cua has one (or two) nitrogens and three (or two) sulphurs with typical distances; these ligands change only slight on reduction. These structures afford the basis for an oxygen reduction mechanism involving oxy- and peroxy intermediates.

Animals↗
PubMed1988

19F-nuclear magnetic resonance: measurements of [O2] and pH in biological systems.

19F-nuclear magnetic resonance (NMR) has been used to determine both intracellular pH and oxygen concentrations in cell suspensions. Oxygen concentrations in Paracoccus denitrificans and insulinoma cells, RINm5F, in the NMR probe can be monitored directly by 1/T1 measurements of perfluorotripropylamine (FTPA)/lecithin emulsion added to the suspensions. With FTPA oxygen monitoring, we investigated the relative aeration capabilities of two types of NMR chambers. Both normal and transformed eucaryotic cells can be maintained in either chamber for at least 1-2 h at cytocrits of up to 20-25%, with 30% oxygen saturation and cell viabilities of 90-95%. Similar concentrations of procaryotes were maintained aerobic with high FTPA concentrations in the more efficient of the two NMR chambers. A new precursor molecule for the 19F-NMR pH indicator difluoromethylalanine, the para-chlorophenyl ester, has been tested and used in RINm5F cells and P. denitrificans, neither of which hydrolyzes methyl esters.

Animals↗
PubMed1999

The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region.

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

Animals↗
PubMed2001

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps.

The cytochrome aa(3)-type quinol oxidase from the archaeon Acidianus ambivalens and the ba(3)-type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme-copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H(+)/electron in a heme-copper oxidase that lacks most residues of the K- and D-channels. Also, the structure of the ba(3) oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D-pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton-pumping mechanism.

Amino Acid Substitution↗
PubMed2002

Replacement of the methionine axial ligand in cytochrome c(550) by a lysine: effects on the haem electronic structure.

The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His-Met cytochromes to His-Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c(550). The electronic structure of the His-Lys haem is shown to be similar to that produced by His-cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand.

Amino Acid Substitution↗
PubMed2002

Inter-subunit cross-linking of methylamine dehydrogenase by cyclopropylamine requires residue alphaPhe55.

Cyclopropylamine is a mechanism-based inhibitor of the quinoprotein methylamine dehydrogenase (MADH) from Paracoccus denitrificans. The resulting inactivation is accompanied by the formation of a covalent cross-link between the alpha and beta subunits of MADH. The results of site-directed mutagenesis studies indicate that Phe55 on the alpha subunit is required for this process. No cross-linking is seen with alphaF55A or alphaF55I MADH mutants. In contrast, with alphaF55E MADH cross-linking of subunits is observed. These results suggest a novel mechanistic role for a phenylalanine residue and the possible importance of protein dynamics in this enzyme mechanism.

Cross-Linking Reagents↗
PubMed2003

Unidirectional effect of lauryl sulfate on the reversible NADH:ubiquinone oxidoreductase (Complex I).

Lauryl sulfate inhibits the Deltamu;(H)(+)-dependent reverse electron transfer reactions catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in coupled bovine heart submitochondrial particles and in vesicles derived from Paracoccus denitrificans. The inhibitor affects neither NADH oxidase (coupled or uncoupled) nor NADH:ferricyanide reductase and succinate oxidase activities at the concentrations that selectively prevent the succinate-supported, rotenone-sensitive NAD(+) or ferricyanide reduction. Possible uncoupling effects of the inhibitor are ruled out: in contrast to oligomycin and gramicidin, which increases and decreases the rate of the reverse electron transfer, respectively, in parallel with their coupling and uncoupling effects, lauryl sulfate does not affect the respiratory control ratio. A mechanistic model for the unidirectional effect of lauryl sulfate on the Complex I catalyzed oxidoreduction is proposed.

Animals↗
PubMed2001

Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase.

The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR). Intensities of the characteristic bands for the high spin heme b, that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (-) in the MCD spectrum decreased considerably. Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g approximately 6, of which less than 5% had been EPR detectable at pH 7. Cyanide (x40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (x1000) was necessary to bind to the reduced NOR at an acidic pH, ca. 5. Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed.

Absorption↗
PubMed1980

Reduction of DL-selenocystine and isolation of L-seleoncysteine.

Cystine, selenocytsine, and several analogs were reduced by dithiothreitol (DTT), beta-mercaptoethanol (ME) and sodium borohydride (NaBH4). DTT was the most effective; DTT to cystine ratios from 10 to 80 were equally effective. With selenocysteine, however, absorption was considerably reduced at all ratios. Selenocysteine was identified as the reduction product by reaction with Gaitonde's reagent, comparison of absorption spectra, paper chromatograhy, utilization by cysteinyl-tRNA synthetase fro Paracoccus denitrificans and Vigna radiata, changes in solubility after DTT treatment, and comparison of infrared spectra. During the ATP-PPi exchange assay, DTT and ME convert cysteine and selenocysteine derivatives to cysteine and selenocysteine which serve as substrates for cysteinyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed2001

Structure of cytochrome c oxidase: a comparison of the bacterial and mitochondrial enzymes.

It has been almost 5 years since the first structures of cytochrome c oxidase, from Paracoccus denitrificans and bovine heart mitochondria, were revealed. Since then many different proton pumping mechanisms have been proposed for the enzyme; however, no definitive conclusion has been achieved. In this article, we revisit the original structures of bacterial and mitochondrial oxidases and try to clarify similarities as well as differences between the two structures.

Animals↗
PubMed2002

Detection, with a pH indicator, of bacterial mutants unable to denitrify.

In order to facilitate isolation of mutants with alterations in the denitrification pathway, a new screening procedure using phenol red incorporated into agar overlay has been defined. Alkalinization in the neighbourhood of denitrifying colonies respiring nitrate or nitrite gives rise to a red circular halo. Antimycin blocked these colour changes, which suggests their association with the periplasmic reduction of nitrite. Inhibition of nitrous oxide reductase by acetylene had no significant effect on alkalinization elicited by nitrate or nitrite. Several mutants negative by the phenol red staining test were generated by transposon Tn5 mutagenesis of Paracoccus denitrificans. All these mutants were defective in the activities of nitrite and nitric oxide reductases while the other denitrification activities were present at the wild-type level.

Acetylene↗
PubMed1997

Potential nitrosamine formation and its prevention during biological denitrification of red beet juice.

High nitrate intake has been shown to result in an increased risk of endogenous formation of N-nitroso compounds. Certain vegetables and vegetable juices contain high concentrations of nitrate. Biological denitrification using strains of Paracoccus denitrificans (P.d.) has been proposed as effective means to reduce nitrate contents in such vegetable juices. During this bacterial denitrification process, substantial nitrite concentrations are transiently formed. This study investigated whether N-nitrosation reactions might occur. The easily nitrosatable amine morpholine was added to red beet juice at high concentration (100 ppm) during denitrification 10 different batches of red beet juice served as raw material. Each batch was submitted to denitrification in the presence and absence of ascorbic acid. In the absence of ascorbic acid, formation of N-nitrosomorpholine (NMOR) was observed in the low ppb range (0.5-8 ppb). Addition of ascorbic acid (500 mg/litre) inhibited the formation of NMOR, except for those instances where the pH was less than 6 and/or nitrate turnover was low (< 200 mg NO3-/litre/hr). Under conditions leading to high rates of nitrate turnover (> 200 mg NO3-/litre/hr), nitrosamine formation can reliably be prevented by ascorbic acid. The results show that bacterial denitrification of red beet juice high in nitrate can be accomplished without the risk of nitrosamine formation.

Ascorbic Acid↗
PubMed1986

Genetics of hydrogenase from aerobic lithoautotrophic bacteria.

Aerobic facultatively autotrophic hydrogen bacteria are distinguished on the basis of their hydrogen-oxidizing enzyme system (Hox). The major group, represented by Paracoccus denitrificans and Pseudomonas facilis, contains a membrane-bound, electron transport-coupled protein. Species of Nocardia are characterized by the possession of a cytoplasmic NAD-dependent hydrogenase. Both enzymes are present in strains of Alcaligenes. All hydrogenases from lithoautotrophs are H2-consuming nickel-iron-sulfur proteins. Despite these common characteristics, hydrogenases differ in catalytic and molecular properties, in particular in the regulation of enzyme synthesis. Hydrogenase formation is either inducible by H2 (e.g. P. denitrificans strain F1, Alcaligenes hydrogenophilus) or subject to derepression in response to the supply of reductant, temperature, and oxygen (e.g. Alcaligenes eutrophus). The only plasmid-encoded Hox function has been conclusively identified in species of Alcaligenes. Structural and regulatory hox genes reside on megaplasmids, ranging in size between 400 and 500 kilobase pairs (kb). Most of the plasmids are self-transmissible by conjugation. Hox genes of A. eutrophus H16 have been localized by plasmid curing, genetic transfer, molecular cloning and analysis of plasmid deletions and insertions. They seem to be clustered in a DNA sequence of approximately 50 kb, representing several transcriptional units. In addition, a chromosomally encoded regulatory function is required for the expression of plasmid-linked hox genes. Plasmid pHGl of A. eutrophus H16 has been transferred to the non-lithoautotrophic soil bacterium JMP222. Both hydrogenases are expressed in the new host. The current state of hydrogenase genetics in Alcaligenes is discussed in reference to hydrogenase systems of other lithoautotrophic bacteria.

Alcaligenes↗
PubMed2003

Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine.

Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water. All strains were in alpha-Proteobacteria. Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria. Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium. Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase. The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.

Aerobiosis↗
PubMed1998

Rhizobium etli cycHJKL gene locus involved in c-type cytochrome biogenesis: sequence analysis and characterization of two cycH mutants.

The cycHJKL gene locus was cloned from Rhizobium etli by the rescue of a Tn5mob insertion of a mutant (IFC01) which was affected in the production of c-type cytochromes. The cycH, cycJ, cycK and cycL genes are proposed to code for different subunits of a haem lyase complex involved in the attachment of haem to cytochrome c apoproteins. CycH of 365 aa shared 27, 36, 47 and 63% identity with CycH from Paracoccus denitrificans, Bradyrhizobium japonicum, R. meliloti, and R. leguminosarum, respectively. CycJ of 153 aa shared 52, 71, and 85% identity to the cycJ gene product of B. japonicum, R. meliloti, R. leguminosarum, respectively. CycK of 666 aa shared 62, 73, and 90% homology with CycK from B. japonicum, R. meliloti, and R. leguminosarum, respectively, while CycL of 151 aa shared 57, 67 and 86% hómology with CycL from the abovementioned species. The Tn5mob insertion present in the IFC01 strain was located in the cycH gene. This strain was able to infect bean plants, but unable to fix nitrogen during symbiosis. A previously described R. etli cytochrome c-deficient MuD1lac-induced mutant (CFN4202) that induced empty nodules on Phaseolus vulgaris, also have lesions in cycH. Complementation analysis suggested that the MuD1lac insertion of the CFN4202 strain was polar on expression of genes downstream of cycH in contrast with the Tn5mob insertion present in IFC01, which showed no polarity on cycJKL. Our data suggest that CycH may not be essential for the infection process, but is necessary for nitrogen fixation.

Bacterial Proteins↗
PubMed1996

Cytochrome c oxidase.

Within the past year, the structures of the cytochrome c oxidase from the soil bacterium Paracoccus denitrificans and of the metal centers of the cytochrome c oxidase from bovine heart mitochondria, both determined at 2.8 A resolution by X-ray crystallography, have been reported. The structures form a basis for understanding the mechanism of this redox-coupled transmembrane proton pump, which is the key component of the respiratory chain of most aerobic organisms.

Animals↗
PubMed2003

Catching catalysis in the act: using single crystal kinetics to trap methylamine dehydrogenase reaction intermediates.

Methylamine dehydrogenase (MADH) is produced by a range of gram-negative methylotrophic and autotrophic bacteria, and allows the organisms to utilise methylamine as the sole source of carbon. The enzyme catalyses the oxidation of methylamine to formaldehyde and ammonia, leaving it in a two-electron reduced state. To complete the catalytic cycle, MADH is reoxidised via an electron transfer (ET) chain. The redox center in the enzyme is the organic cofactor tryptophan tryptophylquinone (TTQ) derived from the posttranslational modification of two Trp residues in the protein. This cofactor has spectral features in the visible region, which change during catalytic turnover, defining spectrally distinct reaction intermediates that reflect the electronic state of the TTQ. In the case of the Paracoccus denitrificans enzyme the physiologic ET chain involves the protein redox partner amicyanin (a blue copper protein). A stable binary (MADH/amicyanin) complex can be formed, and its crystal structure has been solved to 2.5 A resolution by Chen et al. [Biochemistry 21 (1992) 4959]. These crystals were shown to be competent for catalysis and ET by Merli et al. [J. Biol. Chem. 271 (1996) 9177] using single crystal polarised absorption spectroscopy. Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze-trapping, we have trapped reaction intermediates of the enzyme in complex with its physiological redox partner amicyanin in the crystalline state. We will present data confirming that catalysis and ET in the binary complex crystals can be tracked by single crystal visible microspectrophotometry. We will also show that the reaction pathway is unperturbed by the presence of cryoprotectant solution, enabling direct freeze-trapping of reaction intermediates within the crystal. We will present new data demonstrating that the binary complex crystals are also capable of exhibiting UV light-dependent oxidase activity, as observed in solution [Biochim. Biophys. Acta 1364 (1998) 297].

Bacterial Proteins↗