scholarly journals The reaction of fully reduced cytochrome c oxidase with oxygen studied by flow-flash spectrophotometry at room temperature. Evidence for new pathways of electron transfer

1984 ◽  
Vol 218 (3) ◽  
pp. 913-921 ◽  
Author(s):  
B C Hill ◽  
C Greenwood
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Time Course ◽  
Visible Region ◽  
Slow Phase ◽  
Second Phase ◽  
Difference Spectra ◽  
Fast Phase ◽  
O2 Concentration ◽  
Sensitive Phase

Absorption changes during the O2 reaction of reduced bovine cytochrome c oxidase were investigated by the rapid-reaction technique of flow-flash spectrophotometry in the Soret, visible and near-i.r. spectral regions. New features in the time courses of absorption change were observed relative to the earlier findings reported by Greenwood & Gibson [(1967) J. Biol. Chem. 242, 1782-1787]. These new features arise in the Soret and near-i.r. regions and allow the reaction to be described at all wavelengths as a composite of three exponential processes. There is a rapid O2-sensitive phase detectable in the Soret and visible region. The second phase has a rate that is somewhat less dependent on O2 concentration than is the fastest phase rate and is detectable in all three spectral regions. The rate of the third phase is almost independent of the O2 concentration and is also detectable in all spectral regions. Analysis of the three phases gives their rates and absorption amplitudes. The fast phase reaches a rate of 2.5 × 10(4) s-1 at the highest O2 concentration available at 20 degrees C, whereas the phase of intermediate rate is limited at a value of 7 × 10(3) s-1 and the slow phase rate is limited at 700 s-1. The ratios of the kinetic difference spectra for the fast phase and the slow phase do not correspond to the spectra of the individual haem centres. A branched mechanism is advanced that is able to reconcile the kinetic and static difference spectra. This mechanism suggests that some of the cytochrome a is oxidized along with cytochrome a3 in the initial O2-sensitive phase. In addition, the model requires that CuA is oxidized heterogeneously. This fits with the complex time course of oxidation observed at 830 nm while retaining CuA as virtually the sole contributor to absorbance at this wavelength.

scholarly journals Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c

1975 ◽  
Vol 147 (1) ◽  
pp. 145-153 ◽  
Author(s):  
M T Wilson ◽  
C Greenwood ◽  
M Brunori ◽  
E Antonini
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Time Course ◽  
Kinetic Studies ◽  
Initial Reaction ◽  
Fast Phase ◽  
Ferrocytochrome C ◽  
Cytochrome C2 ◽  
Kinetics Of ◽  
Flow Experiments

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with ‘visible’ copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the ‘visible’ copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and ‘visible’ copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.

Fast-phase transvascular fluid flux and the Fahraeus effect

1987 ◽  
Vol 62 (4) ◽  
pp. 1513-1520 ◽  
Author(s):  
W. N. Richardson ◽  
D. Bilan ◽  
M. Hoppensack ◽  
L. Oppenheimer
Keyword(s):  
Mechanical Properties ◽  
Slow Rate ◽  
Time Course ◽  
Rate Of Change ◽  
Distributed Model ◽  
Slow Phase ◽  
Fluid Flux ◽  
Fast Phase ◽  
Constant Rate ◽  
Fahraeus Effect

Transvascular fluid flux was induced in six isolated blood-perfused canine lobes by increasing and decreasing hydrostatic inflow pressure (Pi). Fluid flux was followed against the change in concentration of an impermeable tracer (Blue Dextran) measured directly with a colorimetric device. The time course of fluid flux was biphasic with an initial fast transient followed by a slow phase. Hematocrit changes unrelated to fluid flux occurred due to the Fahraeus effect, and their contribution to the total color signal was subtracted to determine the rate of fast fluid flux (Qf). Qf was related to Pi to derive fast-phase conductance (Kf). Slow-phase Kf was calculated from the constant rate of change of lobe weight. For a mean change in Pi of 7 cmH2O, 40% of the color signal was due to fluid flux. Fast- and slow-phase Kf's were 0.86 +/- 0.15 and 0.27 +/- 0.05 ml X min-1. cmH2O–1 X 100 g dry wt-1. The fast-phase Kf is smaller than that reported for plasma-perfused lobes. Possible explanations discussed are the nature of the perfusate, the mechanical properties of the interstitium, and the slow rate of rise of the driving pressure at the filtration site on the basis of a distributed model of pulmonary vascular compliance.

Cat brain cytochrome-c oxidase redox changes induced by hypoxia after blood-fluorocarbon exchange transfusion

1995 ◽  
Vol 269 (2) ◽  
pp. H417-H424 ◽  
Author(s):  
M. Ferrari ◽  
M. A. Williams ◽  
D. A. Wilson ◽  
N. V. Thakor ◽  
R. J. Traystman ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Redox State ◽  
Near Infrared ◽  
Acute Hypoxia ◽  
Nir Spectroscopy ◽  
Severe Reduction ◽  
O2 Concentration ◽  
O2 Delivery ◽  
Sep Monitoring

We used rapid-scanning near-infrared (NIR) spectroscopy (730-960 nm) to study the effects of graded or acute hypoxia on cerebral cytochrome-c oxidase (cyt aa3) redox state in blood-perfluoro-carbon-exchanged cats with somatosensory evoked potential (SEP) monitoring. In graded hypoxia [10 min each at fractional inspiratory O2 concentration (FIO2) 0.9, 0.8, 0.7, 0.6, and 0.5], cyt aa3 reduction occurred at FIO2 0.6 when cerebral O2 delivery was < 3.5 ml.100 g-1.min-1. In acute hypoxia (FIO2 0.6 for 10 min), significant cyt aa3 reduction occurred from 5 to 10 min (cerebral O2 delivery 3.1 +/- 0.3 ml.100 g-1.min-1) and recovered with reoxygenation (FIO2 1.0). Cyt aa3 redox changes preceded or coincided with SEP alterations in both hypoxia protocols. These results demonstrate that cerebral cyt aa3 reduction occurs with severe reduction of cerebral O2 delivery, but no significant change in cerebral cyt aa3 redox state occurs with small reductions of cerebral O2 delivery. We conclude that substantial changes in cerebral cyt aa3 do not occur at physiological levels of O2 delivery and that current NIR clinical instruments would detect oxygen-dependent cerebral cyt aa3 redox changes only when O2 delivery is extremely compromised.

Pre-steady-state phosphorylation and dephosphorylation of detergent-purified plasma-membrane Ca2+-ATPase

2002 ◽  
Vol 361 (2) ◽  
pp. 355-361 ◽  
Author(s):  
Luis M. BREDESTON ◽  
Alcides F. REGA
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Time Course ◽  
Slow Phase ◽  
Fast Phase ◽  
Lauryl Ether ◽  
Phosphorylation Reaction ◽  
Steady Level ◽  
Biexponential Kinetics ◽  
Acidic Phospholipids

Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca2+-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25°C. The time course of phosphorylation with ATP of the enzyme associated with Ca2+, probably the true phosphorylation reaction, showed a fast phase (kapp near 400s−1) followed by a slow phase (kapp = 23s−1). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (kapp = 93s−1) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (kapp = 30s−1) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (kapp = 220s−1) and slow (kapp = 20s−1) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.

scholarly journals Two Temporal Phases of Light Adaptation in Retinal Rods

2002 ◽  
Vol 119 (2) ◽  
pp. 129-146 ◽  
Author(s):  
Peter D. Calvert ◽  
Victor I. Govardovskii ◽  
Vadim Y. Arshavsky ◽  
Clint L. Makino
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Light Adaptation ◽  
Dynamic Range ◽  
Continuous Illumination ◽  
Slow Phase ◽  
Fast Phase ◽  
Slow Adaptation ◽  
Adaptation Phase

Vertebrate rod photoreceptors adjust their sensitivity as they adapt during exposure to steady light. Light adaptation prevents the rod from saturating and significantly extends its dynamic range. We examined the time course of the onset of light adaptation in bullfrog rods and compared it with the projected onset of feedback reactions thought to underlie light adaptation on the molecular level. We found that adaptation developed in two distinct temporal phases: (1) a fast phase that operated within seconds after the onset of illumination, which is consistent with most previous reports of a 1–2-s time constant for the onset of adaptation; and (2) a slow phase that engaged over tens of seconds of continuous illumination. The fast phase desensitized the rods as much as 80-fold, and was observed at every light intensity tested. The slow phase was observed only at light intensities that suppressed more than half of the dark current. It provided an additional sensitivity loss of up to 40-fold before the rod saturated. Thus, rods achieved a total degree of adaptation of ∼3,000-fold. Although the fast adaptation is likely to originate from the well characterized Ca2+-dependent feedback mechanisms regulating the activities of several phototransduction cascade components, the molecular mechanism underlying slow adaptation is unclear. We tested the hypothesis that the slow adaptation phase is mediated by cGMP dissociation from noncatalytic binding sites on the cGMP phosphodiesterase, which has been shown to reduce the lifetime of activated phosphodiesterase in vitro. Although cGMP dissociated from the noncatalytic binding sites in intact rods with kinetics approximating that for the slow adaptation phase, this hypothesis was ruled out because the intensity of light required for cGMP dissociation far exceeded that required to evoke the slow phase. Other possible mechanisms are discussed.

scholarly journals Circular-dichroic properties and secondary structure of Pseudomonas aeruginosa soluble cytochrome c oxidase

1984 ◽  
Vol 218 (3) ◽  
pp. 907-912 ◽  
Author(s):  
M G Tordi ◽  
M C Silvestrini ◽  
A Colosimo ◽  
S Provencher ◽  
M Brunori
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Secondary Structure ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Visible Region ◽  
Alpha Helix ◽  
Careful Analysis ◽  
Amino Acid Residues ◽  
Ferrocytochrome C ◽  
Significant Difference

The c.d. spectra of Pseudomonas aeruginosa cytochrome c oxidase in the oxidized state and the reduced state are reported in the visible- and u.v. absorption regions. In the visible region the comparison between the spectra of reduced cytochrome c oxidase and ferrocytochrome c-551 allows the identification of the c.d. bands mainly due to the d1 haem chromophore in cytochrome c oxidase. In the near-u.v. region the assignment of some of the observed peaks to the haem groups and to the aromatic amino acid residues is proposed. A careful analysis of the data in the far-u.v. region leads to the determination of the relative amounts of alpha-helix and beta-sheet in the enzyme, giving for the first time a picture of its secondary structure. A significant difference in this respect between the reduced and the oxidized species is observed and discussed in the light of similar conclusions reported by other workers.

scholarly journals Operando Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

2020 ◽  
Author(s):  
Sven Timo Stripp
Keyword(s):  
Infrared Spectroscopy ◽  
Cytochrome C ◽  
Ambient Temperature ◽  
Cytochrome C Oxidase ◽  
Structural Changes ◽  
Attenuated Total Reflection ◽  
Invasive Technique ◽  
Difference Spectra ◽  
Gas Processing ◽  
Temperature And Pressure

Earth-abundant transition metals like iron, nickel, copper, molybdenum, and vanadium have been identified as essential constituents of the cellular gas metabolism in all kingdoms of life. Associated with biological macromolecules, gas-processing metalloenzymes (GPMs) are formed that catalyse a variety of redox reactions. This includes the reduction of O2 to water by cytochrome c oxidase (&lsquo;complex IV&rsquo;), the reduction of N2 to NH4 by nitrogenase, as well as the reduction of protons to H2 (and oxidation of the later) by hydrogenase. GPMs perform at ambient temperature and pressure, in the presence of water, and often extremely low educt concentrations, thus serving as natural examples for efficient catalysis. Facilitating the design of biomimetic catalysts, biophysicist thrive to understand the reaction principles of GPMs making use of various techniques. In this perspective, I will introduce Fourier-transform infrared spectroscopy in attenuated total reflection configuration (ATR FTIR) for the analysis of GPMs like cytochrome c oxidase, nitrogenase, and hydrogenase. Infrared spectroscopy provides information about the geometry and redox state of the catalytic cofactors, the protonation state of amino acid residues, the hydrogen-bonding network, and protein structural changes. I developed an approach to probe and trigger the reaction of GPMs by gas exchange experiments, exploring the reactivity of these enzymes with their natural reactants. This allows recording sensitive ATR FTIR difference spectra with seconds time resolution. Finally yet importantly, infrared spectroscopy is an electronically non-invasive technique that allows investigating protein samples under biologically relevant conditions, i.e., at ambient temperature and pressure, and in the presence of water.

The Kinetics of Sodium Transfer in the Central Nervous System of the Cockroach, Periplaneta Americana L

1961 ◽  
Vol 38 (4) ◽  
pp. 737-746
Author(s):  
J. E. TREHERNE
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Periplaneta Americana ◽  
Slow Phase ◽  
Second Phase ◽  
Sodium Ions ◽  
Rapid Phase ◽  
Fast Phase ◽  
The Central Nervous System ◽  
Sodium Extrusion

1. The exchange of sodium ions in the cockroach central nervous system has been studied by following the escape of 24Na from isolated abdominal nerve cords, single connectives and ganglia. Particular attention was paid to the initial rapid exchanges of sodium. 2. The escape of sodium ions occurred as a two-stage process, an initial rapid phase eventually giving way to a slower exponential phase of sodium loss. The fast phase of efflux was not affected by the presence of 2:4-dinitrophenol, although this poison significantly reduced the second slow phase of sodium extrusion. 3. The initial fast phase is attributed to a rapid diffusion from an extracellular space, demonstrated by 14C-inulin; the second phase is identified as the slower extrusion from the cellular components of the central nervous system.

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