Bacterium Paracoccus Denitrificans Research Articles

Cytochrome c1 from Paracoccus denitrificans.

Cytochrome c1 was purified from the bacterium Paracoccus denitrificans. It is an acidic, hydrophobic polypeptide with an apparent molecular weight of around 65000 and a single, covalently attached heme; it cross-reacts immunologically with cytochrome c1 from yeast mitochondria. The amino acid sequence of the tryptic heme peptide of the bacterial cytochrome c1 shows extensive homology to the corresponding region of beef heart cytochrome c1 [Wakabayashi, S. et al. (1982) J. Biol. Chem. 257, 9335-9344]. Positive evidence for a stable association of the Paracoccus cytochrome c1 with other polypeptides and b-type heme components ('bc1-complex') has not yet been obtained.

Solubilized cytochrome c oxidase from Paracoccus denitrificans is a monomer.

Cytochrome c oxidase purified from the bacterium Paracoccus denitrificans was analyzed by analytical ultracentrifugation. In the detergent octyltetra/pentaoxyethylene (C8E45), the isolated enzyme exhibits a molecular weight of 79,000 to 84,000. The detergent-solubilized enzyme is thus a monomer which contains one copy of each of the two subunits.

Proton-coupled energy transduction by biological membranes. Principles, pathways and praxis

A brief outline of certain features of the chemiosmotic hypothesis of the mechanism of free-energy transfer between the reactions of electron transport and adenosine triphosphate synthesis catalysed by biological membranes is given. Pulses of electron transport induced by the addition of small quantities of oxygen to suspensions of the bacterium Paracoccus denitrificans lead to vectorial H+ movements into the aqueous phase external to the organisms, where they may be detected with a glass pH electrode. The stoichiometry of the number of protons translocated into the bulk phase external to the organisms, per oxygen atom reduced, is essentially unchanged when the amount of oxygen reduced is varied, in a manner inconsistent with the predictions of the chemiosmotic-coupling hypothesis. These and other observations lead to the view that the energy-coupling proton-transfer processes utilised in reactions such as electron-transport phosphorylation are confined to the membrane phase. Mechanisms which most easily account for this are discussed.

Resonance Raman spectra of cytochromes c and b in Paracoccus denitrificans membranes: evidence for heme--heme interactions.

Resonance Raman (RR) scattering from cytochromes b and c in the bacterium Paracoccus denitrificans was recorded by exciting with the 568.2-, 530.9-, and 520.8-nm lines of a Kr+ laser. The main features of the spectra were similar to those of the analogous cytochrome b-c1 complex derived from pigeon breast mitochondria. Differences in the 1300-cm-1 region were interpreted in terms of marker bands for heme type and spectral coupling of the hemes on the membranes. It is difficult to explain the results without invoking sharing of electronic and vibrational wave functions among the hemes. This conclusion documents the potential to study the physics of electron transport in functioning membrane by monitoring the RR spectra.

Control of nitrate respiration inParacoccus denitrificansby oxygen

The denitrifying bacterium Paracoccus denitrificans possesses a constitutive respiratory chain, resembling closely its mammal i an mitochondrial counterpart, a part of which is functional in the electron transfer to nitrate under anaerobic conditions of growth ([1] and refs. therein). The terminal part of the anaerobic electron transfer is represented by the membrane bound nitrate reductase. A characteristic feature of cells grown on nitrate is that the dissimilatory reduction of nitrate is strongly inhibited by 02 [2]. The effect of 02 was observed also when working with spheroplasts [3]. By contrast, cells treated with Triton X-100 and plasma membrane vesicles reduced nitrate and oxygen simultaneously [2,3]. The results were interpreted as indicating that access of nitrate to the active site of its reductase was prevented by oxygen ([3], but see [4]). In the present study we have obtained evidence that oxygen exerts its controlling effect by changing the redox state of membrane components via terminal oxidases.

A reassessment of the structure of Paracoccus cytochrome c-550

An amino acid sequence and a three-dimensional structure of cytochrome c-550 from the facultatively denitrifying aerobic bacterium Paracoccus denitrificans have been reported (Timkovich et al., 1976; Timkovich & Dickerson, 1976). The amino acid sequence showed considerable similarity to Rhodospirillaceae (purple phototrophic bacterial) cytochrome c 2, but also had some unexpected features. We have reexamined the amino acid sequence and have found five discrepancies. The molecule contains an additional tryptophan residue, which was not detected in either the 2.5 Å crystallographic analysis or the original sequence investigation.

Production of superoxide anions in Paracoccus denitrificans

Like mitochondria, plasma membranes of the free-living bacterium Paracoccus denitrificans are able to produce superoxide ions. The production of superoxide ions was observed during the initial stages of electron transfer from the respiratory substrates to oxygen, even when the bacteria had been grown anaerobically on nitrate as oxidant. Generation of Superoxide anions was supported by NADH or succinate and occurred before the antimycin-sensitive site of the respiratory chain, presumably at the level of a low potential redox component. Superoxide anion formation was pH and substrate dependent; it was inhibited by cyanide and by exogenous superoxide dismutase.

The orientation of cytochromes in membrane multilayers prepared from aerobically grown Escherichia coli K12.

Centrifugation of membrane vesicles, prepared from ultrasonically disrupted Escherichia coli K12, on to a planar surface followed by slow, partial dehydration results in a high degree of parallel orientation of the membrane planes with respect to each other and the supporting surface. Rotation of such membrane multilayers about a single axis parallel with the membrane planes within the magnetic field of an electron paramagnetic resonance (e.p.r.) spectrometer allows the orientation of anisotropic paramagnetic centres to be deduced. Computer simulations of the angular dependence of cytochrome e.p.r. spectra show two, or perhaps three, cytochromes, well-oriented with respect to the membrane plane. A low-spin cytochrome is oriented with the normal to its haem plane lying in the membrane plane. One (or perhaps two) high-spin cytochrome(s) lies with its haem plane making an angle of 45 degrees with the membrane plane. The orientation of the low-spin cytochrome haem is thus the same as that of haems in b-type cytochromes and cytochrome oxidases of the a type found in the mitochondria of higher animal and microbial cells and the bacterium Paracoccus denitrificans (Erecińska et al., 1979). The possible identity of this low-spin component as the terminal oxidase, cytochrome o, is discussed.

A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans.

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) was purified from the cytoplasmic membrane of the bacterium Paracoccus denitrificans. The enzyme contains two heme groups (a and a3) and two copper atoms per minimal unit, oxidizes mammalian cytochrome c at a high rate, and, when incorporated into liposomes, generates an electrochemical proton gradient during cytochrome c oxidation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals only two subunits of apparent molecular weights 45,000 and 28,000; they appear to correspond to the two largest mitochondrially made subunits of the seven-subunit cytochrome c oxidase isolated from yeast mitochondria. Because of its structural simplicity. Paracoccus cytochrome c oxidase offers new possibilities for exploring the mechanism of cytochrome c oxidase function.