This article reviews the significance of the subunit structure of cytochrome-c oxidase in proton pumping and in particular summarizes available evidences for or against a role of subunit III in the control of this important function of the enzyme.

Chemical Phenomena↗

PubMed1987

Modulation of cytochrome oxidase activity by inorganic and organic phosphate.

The activity of cytochrome oxidase reconstituted into phospholipid vesicles has been studied as a function of orthophosphate, ATP and inositol hexakisphosphate concentrations. The respiratory-control ratio was found to be quite sensitive to these compounds and was inversely related to the anion concentration. This effect is related to a phosphate-dependent decrease in the rate constant for ferrocytochrome c oxidation observed in the presence of ionophores. The data cannot be interpreted simply on the basis of ionic strength, which is known to limit cytochrome c binding to cytochrome oxidase, since cytochrome oxidase-containing vesicles responded differently to phosphate depending on the energization state of the phospholipid membrane.

Adenosine Triphosphate↗

PubMed1987

Xenopus laevis hemoglobin and its hybrids with hemoglobin A+.

Isolated alpha and beta chains from Xenopus laevis hemoglobin have been purified. The isolation procedure yields native alpha chains whose functional behavior has been characterized and compared with that of human alpha chains. Isolated beta chains in the presence of oxygen are characterized by low stability, and hence their functional characterization was limited to the CO binding kinetics. When stoichiometric amounts of the isolated alpha and beta chains are mixed, a tetramer characterized by heme-heme interactions and oxygen affinity comparable to that of the native molecule is readily reconstituted. Moreover, both chains, under appropriate conditions, form stable hybrid tetramers with the partner subunits from human hemoglobin; results on the functional properties of these hybrid hemoglobins are presented and discussed in relation to the stereochemical model of the Root effect.

Animals↗

PubMed1987

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies.

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.

Animals↗

PubMed1987

Haem disorder in two myoglobins: comparison of reorientation rate.

The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237, 613-616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon.

Animals↗

PubMed1987

Glutathione peroxidase and oxidative hemolysis in trout red blood cells.

Red blood cells from the trout Salmo irideus contain several hemoglobin components that are prone to oxidation with production of oxygen radicals. The rate of hemolysis has been correlated to the extent of methemoglobin formation. A difference in the rate of hemolysis between red blood cells saturated with either CO or O2 was evident only when diminished glutathione peroxidase activity was observed. These results confirm the important role of this enzyme in providing protection against or repair of oxidative damage to the red cell membrane.

Animals↗

PubMed1987

Is there a Root effect in Xenopus hemoglobin?

The reaction of Xenopus hemoglobin with oxygen and carbon monoxide has been reinvestigated over the pH range 8.5-6.0, in the absence and presence of organic phosphates (2,3-diphosphoglycerate or inositol hexakisphosphate), to establish if the tetramer can be stabilized in a T-quaternary state by protons and polyphosphate; the equilibrium and kinetic data indicate that Xenopus hemoglobin does exhibit a Root effect. These new results are discussed with reference to those reported by Bridges et al. [(1985) Resp. Physiol. 61, 125-136] on Xenopus blood and, more generally, to the molecular definition and the structural basis of the Root effect as an extreme form of the Bohr effect.

2,3-Diphosphoglycerate↗

PubMed1987

Unfolding and flexibility in hemoproteins shown in the case of carboxymethylated cytochrome c.

A circular dichroism study of carboxymethylated cytochrome c has been performed to obtain further information on the structural basis responsible for the observed changes in ligand binding and redox properties of the modified cytochrome c. The results give additional evidence of local structural changes occurring in the heme environment upon rupture of the (Met-80)-heme iron bond in the modified protein. This produces no alterations of the overall molecular conformation, but results in drastic changes in redox potential. In addition, analysis of the reversible conformational transitions induced by urea in the native and the modified proteins supports the idea that the modified derivative can be considered as an 'intermediate state' between the native and the fully unfolded protein.

Animals↗

PubMed1987

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units.

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.

Animals↗

PubMed1987

Human hemoglobin cross-linked through the polyphosphate-binding site. Functional properties and evidence for conformers.

The properties of human hemoglobin reacted with 2-nor-2-formylpyridoxal 5'-phosphate, a bifunctional derivative of pyridoxal 5'-phosphate, have been investigated both from an equilibrium and kinetic point of view. The experimental data, interpreted in terms of the two-state allosteric model, indicate that a perturbed R state is characteristic of this modified low ligand affinity hemoglobin. In flash photolysis experiments, a quickly reacting component is always observed, in spite of the lack of dissociation into free dimers; this kinetic behavior is thought to reflect the presence of functionally independent alpha beta dimers, still connected by the flexible cross-link but forming an open hemoglobin tetramer. Two possible models for the interpretation of the kinetics of CO and/or haptoglobin binding are presented and discussed.

Hemoglobins↗

PubMed1987

Kinetics of Pseudomonas aeruginosa cytochrome c551 and cytochrome oxidase oxidation by Co(phen)3(3+) and Mn(CyDTA)(H2O)-.

The reaction between reduced Pseudomonas cytochrome c551 and cytochrome oxidase with two inorganic metal complexes, Co(phen)3(3+) and Mn(CyDTA)(H2O)-, has been followed by stopped-flow spectrophotometry. The electron transfer to cytochrome c551 by both reactants is a simple process, characterized by the following second-order rate constant: k = 4.8 X 10(4) M-1 sec-1 in the case of Co(phen)3(3+) and k = 2.3 X 10(4) M-1 sec-1 in the case of Mn(CyDTA)(H2O)-. The reaction of the c-heme of the oxidase with both metal complexes is somewhat heterogeneous, the overall process being characterized by the following second-order rate constants: k = 1.7 X 10(3) M-1 sec-1 with Co(phen)3(3+) and k = 4.3 X 10(4) M-1 sec-1 with Mn(CyDTA)(H2O)- as oxidants; under CO (which binds to the d1-heme) the former constant increases by a factor of 2, while the latter does not change significantly. The oxidation of the d1-heme of the oxidase by Co(phen)3(3+) occurs via intramolecular electron transfer to the c-heme, a direct bimolecular transfer from the complex being operative only at high metal complex concentrations; when Mn(CyDTA)(H2O)- is the oxidant, the bimolecular oxidation of the d1-heme competes successfully with the intramolecular electron transfer.

Bacterial Proteins↗

PubMed1987

Fluorescence and energy transfer of tryptophans in Aplysia myoglobin.

The fluorescence decay of tryptophan residues in apo and met Aplysia limacina myoglobin and sperm whale myoglobin were measured in aqueous solution at 10 degrees-15 degrees C. In all species, multiexponential behavior was observed in which the individual components displayed unique frequency-dependent emission characteristics. The results suggest that the tryptophan fluorescence in all met samples are quenched by rapid Forster energy transfer to the heme as predicted from the crystal geometry. Fluorescence from the apo protein is similar to that in solutions of free tryptophans. In addition, the fluorescence properties of the reversible thermal denaturation of Aplysia limacina met myoglobin was investigated between 25 degrees and 75 degrees C.

Animals↗

PubMed1987

A microspectroscopic analysis of ligand binding in single erythrocyte.

The kinetics of ligand uptake by erythrocytes has been analyzed by single cell microspectroscopy measurements carried out in parallel with mathematical simulations. The main experimental feature is the linear shape of the ligand binding progress curves, whose slope is dependent on the free ligand concentration in the speciman. From the computer simulation a predominant role has been shown to be played by the ligand diffusion from the extracellular space.

Carbon Monoxide↗

PubMed1986

Evidence for the interaction between the calcium indicator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and calcium-binding proteins.

Stopped-flow and static difference spectroscopy experiments have shown that the calcium indicator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) interacts with several different calcium-binding proteins (beta-trypsin, parvalbumin, and calmodulin) and with serum albumin under experimental conditions commonly used in biophysical studies. The interaction decreases at high ionic strength. EDTA competes with BAPTA in the interaction with the proteins.

Animals↗