Electron self-exchange kinetics in two average-valence dicopper cryptates

Electron self-exchange rates, reported for two average-valence dicopper cryptates are, at around 10(5) M-1 s-1, at the high end of the range for small-molecule model compounds. The cross-exchange reaction rate, and those for oxidation with [Co(ox)3]3-, are consistent with an outer-sphere reaction mechanism. Despite the necessity for copper-copper bond making and breaking in the course of redox for one of the cryptates, the self-exchange rate is not decreased relative to the other cryptate, showing that this step is not rate limiting.     

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.

The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.     

Electron transport and cytochromes in aerobically grown Proteus mirabilis

The function of the cytochromes in electron transport from NADH to oxygen in aerobically grown Proteus mirabilis has been determined. 77K-Spectra of cytoplasmic membrane suspensions, frozen while catalyzing electron transport from NADH to oxygen, in the presence as well as in the absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide, have been recorded. Analysis of these 77K-spectra revealed that cytochrome b-563 (E'0 = +140 mV), cytochrome b-556 (E'0 = +140 mV) [or alternatively cytochrome b-563/556 (E'0 = +140 mV)] and cytochrome b-557 (E'0 = +50 mV) may function in a Q or b-cycle. The function of cytochrome c-549 (E'0 = +75 mV), which seems to be present only in a very low concentration, and cytochrome b-556 (E'0 = -105 mV), which reacts very slowly to the addition of NADH and oxygen, remains unclear. Cytochrome o, the main oxidase of aerobically grown P. mirabilis cells, can not be detected by the methods described above. Only when the reduced form of cytochrome o is liganded with carbon monoxide a specific alpha-band can be detected at 569 nm at 25 degrees C and 565 nm at 77K.     

Electron transport in cytochromes P-450 by covalent switching.

The mechanism of electron transfer in cytochrome P-450cam is presented in terms of a covalent switching mechanism. We present a model of putidaredoxin built by homology, which helps explain protein-protein interactions. The mechanism is general enough to account for the genetic variations found in the superfamily of cytochromes P-450. The detail should assist in the design of novel P-450 inhibitors and may have wider implications. The sequence analysis supports our protein model, and highlights the role of cystein and aromatic residues in electron-transport mechanisms. Eukaryotic cytochromes P-450 appear to have evolved their own intramolecular tryptophan electron-transfer mediator, unlike prokaryotic P. putida P-450cam, which still relies upon the C-terminal tryptophan of its attendant electron-transport protein, putidaredoxin. On this basis our protein model is capable of rationalizing the transfer of electrons from NADH to the active site of P-450. At the electronic level the covalent switching that transfers pairs of electrons not only provides a plausible mechanism, but may also have ramifications in a wider context.     

Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas

Two cytochromes c₅ (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c₅₅₁ (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.     

The two cytochromes c in the facultative methylotroph Pseudomonas AM1

It was previously suggested that there is only one soluble cytochrome c in Pseudomonas AM1, having a molecular weight of 20000, a redox midpoint potential of about +260mV and a low isoelectric pint [Anthony (1975) Biochem. J. 146, 289–298; Widdowson & Anthony (1975) Biochem. J. 152, 349–356]. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 has now revealed the presence of two different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isoelectric focusing. Molecular weights were determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behaviour of the two cytochromes on ion-exchange celluloses. The more abundant cytochrome c_H (λ_max. 550.5nm) had a low molecular weight (11000), a midpoint potential of about +294mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The less abundant cytochrome c_L (λ_max. 549nm) was about 30% of the total; it had a high molecular weight (20900), a midpoint potential of about +256mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The pH-dependence of the midpoint redox potentials of the two cytochromes c were very similar. There were four ionizations affecting the redox potentials in the pH range studied (pH4.0–9.5), two in the oxidized form (pK values about 3.5 and 5.5) and two in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the two propionate side chains of the haem. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C₁ compounds, although still able to grow well on multicarbon compounds such as succinate. Whether or not these two cytochromes c have separate physiological functions is not yet certain.     

Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins

Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins. Cu(I) to "Re(II)" electron tunneling in Re(H107) azurin occurs in the microsecond range. This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep ("hopping") mechanism might be involved. Although a Y108 radical can be generated by flash-quenching a Re(H107)M(II) (M=Cu, Zn) protein, the evidence suggests that it is not an active intermediate in the enhanced Cu(I) oxidation. Rather, the likely explanation is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radical), followed by electron tunneling from Cu(I) to the hole in the imidazole ligand.     

Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp

 A comparative study of electron transfer between the 16 heme high molecular mass cytochrome (Hmc) from Desulfovibrio vulgaris Hildenborough and the [Fe] and [NiFe] hydrogenases from the same organism was carried out, both in the presence and in the absence of catalytic amounts of cytochrome c ₃. For comparison, this study was repeated with the [NiFe] hydrogenase from D. gigas. Hmc is very slowly reduced by the [Fe] hydrogenase, but faster by either of the two [NiFe] hydrogenases. In the presence of cytochrome c ₃, in equimolar amounts to the hydrogenases, the rates of electron transfer are significantly increased and are similar for the three hydrogenases. The results obtained indicate that the reduction of Hmc by the [Fe] or [NiFe] hydrogenases is most likely mediated by cytochrome c ₃. A similar study with D. vulgaris Hildenborough cytochrome c ₅₅₃ shows that, in contrast, this cytochrome is reduced faster by the [Fe] hydrogenase than by the [NiFe] hydrogenases. However, although catalytic amounts of cytochrome c ₃ have no effect in the reduction by the [Fe] hydrogenase, it significantly increases the rate of reduction by the [NiFe] hydrogenases. Received: 14 April 1998 / Accepted: 25 June 1998     

椰毒假单脆菌的电镜观察

A strain of food-poisoning bacterium has been isolated by Jin Jiexiang (1963) in China from the fermented cornflour that has gone bad. These pathogenic microorganism has been identified and named Pseudomonas by Zhao Naixin in 1988, which is the same species as P. cocovenenans. The characteristics of them were conformed to these of the species P. cepacia of section 2 of the genus Pseudomonas. In view of the fact that the fine structures of the above mentioned three strains of Pseudomonas have not been described yet, we decided to observe them with electron microscope. Results indicate there are many things in common among the three strains, such as: appearing short rods, 0.6-0.8 microns in diameter by 1.5-2.0 microns in length, one polar multiflagella; non-pili, non-capsules, non-endospores; containing intranuclear inclusions (electron-dense bodies or concentric laminae bodies), accumulating intracytoplasmic PHB granules; forming filaments, minicells and bizarrecells; producing extracellular cellulose-like materials by the three strains have not been reported previously.     

The respiratory system of Pseudomonas putida: participation of cytochromes in electron transport

Rates of oxygen utilization by Pseudomonas putida respiratory particles were measured using the electron donors, reduced nicotinamide adenine dinucleotide (NADH) and succinate, and the oxidation-reduction dyes, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethyl-p-phenylenediamine. The maximal rates produced by NADH and succinate were similar for particles from either log- or stationary-phase cells, but rates measured using the dyes were much higher in stationary-phase particles. Cyanide and azide were very effective inhibitors of dye oxidation in both cases, but they produced only partial inhibition of NADH and succinate oxidation in log-phase particles and had no effect in the stationary phase. Spectral examination of the cytochromes at several levels of reduction produced by the various electron donors and inhibitors indicated that most of the cytochromes that were reduced by the dyes lie on a cyanide sensitive pathway of electron transport. These findings support the hypothesis that P. putida produces an electron transport system in the stationary phase which involves branching at the level of the cytochromes.Inhibition of oxygen utilization by CO was nearly complete for all four substrates in logphase particles. Inhibition was also reasonably effective for dye oxidation in the stationary phase, but there was no effect on NADH or succinate oxidation. Photochemical action spectra of the relief of CO inhibition revealed that NADH and succinate oxidation in log-phase particles probably involves cytochrome o. Oxidation of the dyes by either type of particles also appeared to involve cytochrome o, and the possibility of the participation of an a- or d-type cytochrome was also indicated.     

Electron transfer reactions of high-potential cytochromes in the reaction centre of Chromatium tepidum

In the thermophilic purple bacterium C. tepidum, the reaction centre (RC) has a bound cytochrome, containing two high-potential hemes (E_m above +350 mV) and two low-potential hemes (E_m below +150 mV), which re-reduces the photooxidized primary donor, P⁺. We have studied the effects of ambient redox potential and of temperature on the kinetics of that reaction by kinetic flash absorption spectroscopy in chromatophores and isolated reaction centers. When both high-potential hemes are reduced prior to excitation by a short flash of light, the halftime increases slightly between 294 K (t_1/2 = 500 ns) and 217 K (t_1/2 = 1040 ns) indicating an activation energy of 5.0 kJ mol^–1. The fraction of P⁺ which decays by this fast reaction decreases rather steeply around 220 K from nearly 100% at 294 K to nearly 0% below 190 K where P⁺ decays slowly (t_1/2 2.5 ms), probably by return of an electron from the quinone acceptors. When the high-potential hemes are partially oxidized prior to the flash, an additional kinetic phase having a halftime of 30 µs at 294 K is observed. The fractions of RCs that give rise to the individual kinetic phases of P⁺ reduction have been monitored as a function of redox potential. The results can be interpreted in terms of two high-potential hemes which have similar midpoint potentials of +380 ±10 mV and a weak electrostatic interaction.     

Half reduction potentials and oxygen affinity of the cytochromes of Pseudomonas carboxydovorans

Abstract The midpoint redox potentials (E'₀) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H₂+ CO₂; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO-sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b -555, about −20 mV; cytochrome c and cytochrome a , about +220 mV). For the cytochromes of the CO-insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b -558) and −11 to −215 mV (cytochrome b -561) were determined. Cytochrome [ib-561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent K _m value for oxygen revealed a low affinity of cytochrome a ( K _m/ 5 υ M O₂) and a very high affinity of the CO-insensitive oxidase ( K _m < 0.5 μ M O₂). The high affinity to oxygen might be responsible for the CO-insensitivity of this unusual cytochrome o .     

Electron Microscopy of Pseudomonas φ6 Bacteriophage

phi6 bacteriophage from Pseudomonas phaseolicola has a pleomorphic shape due to an outer layer of lipid. This layer was removed with organic solvents or sodium dodecyl sulfate revealing a 50-nm cubic particle. The virus was found only in the central portion of the cell and not near the cell wall.