Cytochrome c oxidase: Intermediates of the catalytic cycle and their energy-coupled interconversion

Several issues relevant to the current studies of cytochrome c oxidase catalytic mechanism are discussed. The following points are raised. (1) The terminology currently used to describe the catalytic cycle of cytochrome oxidase is outdated and rather confusing. Presumably, it would be revised so as to share nomenclature of the intermediates with other oxygen-reactive heme enzymes like P450 or peroxidases. (2) A "catalytic cycle" of cytochrome oxidase involving complete reduction of the enzyme by 4 electrons followed by oxidation by O(2) is a chimera composed artificially from two partial reactions, reductive and oxidative phases, that never operate together as a true multi-turnover catalytic cycle. The 4e(-) reduction-oxidation cycle would not serve a paradigm for oxygen reduction mechanism and protonmotive function of cytochrome oxidase. (3) The foremost role of the K-proton channel in the catalytic cycle may consist in securing faultless delivery of protons for heterolytic O-O bond cleavage in the oxygen-reducing site, minimizing the danger of homolytic scission reaction route. (4) Protonmotive mechanism of cytochrome oxidase may vary notably for the different single-electron steps in the catalytic cycle.     

Electrogenic reactions of cytochrome bd

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge.     

Conversion of Natural and Associated Petroleum Gases in Cold Electron-Beam Plasma

A device is developed to create cold nonequilibrium electron-beam plasma in a supersonic gas flow. The possibility of conversion of natural and associated petroleum gases into products with different chemical compositions by using this plasma is demonstrated. With the use of laboratory equipment, we find various products of oxidative and nonoxidative conversion. The proposed method is promising for industrial application.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

In-situ microscopy: Online process monitoring of mammalian cell cultures

The in-situ microscope is a system developed to acquire images of mammalian cells directly inside a bioreactor (in-situ) duringa fermentation process. It requires only minimal operator intervention and it is well suited for either batch or long-termperfusion fermentation runs. The system fits into a 25 mm standard port and has a retractable housing, similar to the industry standard InTrac. Therefore, it can be cleaned and serviced without interruption of the process or risking contamination. A sampling zone inside the bioreactor encloses adefined volume of culture and an image sequence is taken. The height of the sampling zone is set by the control program and canbe adjusted during the cultivation to accommodate a wide range of change in cell density. The system has an infinity correctedoptical train and uses a progressive scan CCD camera to acquirehigh quality images. Process relevant information like cell density is extracted fromthe images by digital image processing software, currently in development for mammalian cells (CHO, BHK). The first version ofthe software will be able to estimate the cell density, cellsize distribution and to give information of the degree of aggregation (single and double cells, cell clusters).     

Fermentor temperature as a tool for control of high-density perfusion cultures of mammalian cells

Temperature is a key environmental variable whose potential in animal cell fermentor optimization is not yet fully utilized. The scarce literature data suggests that reduced fermentor temperature results in an improved viability and shear resistance, higher cell density and titer in batch cultures, and reduction in glucose/lactate metabolism. Due to the arrest of the cells in the G1 phase, the specific growth rate was found to decrease at temperatures below 37.0 degrees C. The response of the specific production rate was cell line dependent: in some cases it increased 2-to-3-fold, but decreased in other cases. The controlable slowdown of cell metabolism at lower temperature can be used in optimization of perfusion mammalian cell cultures with several potential advantages, including higher cell density in oxygen limited reactors, lower perfusion rate, improved product quality, simplified pH control, and others. To evaluate this strategy, a series of long-term experiments in 15 L perfusion bioreactors culturing recombinant hamster cells at 20.0 x 10(6) cells/mL were conducted. The temperature was changed over a range of set points, and maintained at each of these for a long period of time. Steady state process data was collected and analyzed. The effect of temperature on the following characteristics of the perfusion process was studied: cell growth, glucose/lactate metabolism, glutamine/ammonia metabolism, cell respiration, cell density at constant oxygen transfer rate, proteolytic activity, and product quality (glycosylation and molecule fragmentation). The results suggest that temperature is a variable with a significant potential in optimization of perfusion cultures. Properly selected temperature set point will contribute to the overall improvement of process performance. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 328-338, 1997.