Karin M.C. Sinjorgo

The effects of pH and ionic strength on cytochrome c oxidase steady-state kinetics reveal a catalytic and a non-catalytic interaction domain for cytochrome c.

The influence of pH and ionic strength on the steady-state kinetics of purified bovine cytochrome c oxidase was studied by spectrophotometry. At low ionic strength, increasing the pH in the range between 5.4 and 8.6 resulted in a slight decrease in maximal turnover numbers of the high-affinity and the low-affinity reactions. The high-affinity Km was also found to decrease with increasing pH. The ionic-strength dependence of the steady-state kinetics of positively charged cytochrome c oxidase at pH 6.2 and that of negatively charged cytochrome c oxidase at pH 7.8 were similar; in both cases, high-affinity Km values and high-affinity and low-affinity TNmax values increased with ionic strength. The low-affinity Km was independent of both pH and ionic strength. Above I = 100 mM, no low-affinity reaction could be observed. A description of the electrostatic interactions between cytochrome c and cytochrome c oxidase, based on the overall monopoles and overall dipoles of the two proteins, could not explain our data. We propose that at I greater than or equal to 25 mM such an approximation cannot be used for electrostatic interactions between large proteins, since the assumption that all charges on the surfaces of the reacting proteins would contribute equally to the electrostatic interaction is not valid. A qualitative description of electrostatic interactions between the two cytochromes based on limited electrostatic interaction domains on the cytochrome c oxidase surface was found to be in good agreement with all our data and supports the model of Speck et al. (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 347-351), who proposed one catalytic and one non-catalytic cytochrome c binding site. It is proposed that the allosteric effect of the cytochrome c at the non-catalytic site is of an electrostatic nature. At high ionic strength (occurring in vivo), this cytochrome c molecule would then no longer affect the catalytic site, resulting in the absence of the low-affinity reaction.

Bovine cytochrome c oxidases, purified from heart, skeletal muscle, liver and kidney, differ in the small subunits but show the same reaction kinetics with cytochrome c

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.

The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics

(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots (Km congruent to 1.10(-8) M and Km congruent to 2.10(-5) M). It is not necessary to postulate a third reaction of Km congruent to 10(-6) M. (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed.

Human cytochrome c oxidase isoenzymes from heart and skeletal muscle; purification and properties

Human cytochrome c oxidase was isolated in an active form from heart and from skeletal muscle by a fast, small-scale isolation method. The procedure involves differential solubilisation of the oxidase from mitochondrial fragments by laurylmaltoside and KCl, followed by size-exclusion high-performance liquid chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed differences between the subunit VI region of cytochrome c oxidases from human heart and skeletal muscle, suggesting different isoenzyme forms in the two organs. This finding might be of importance in explaining mitochondrial myopathy which shows a deficiency of cytochrome c oxidase in skeletal muscle only. In SDS polyacrylamide gel electrophoresis most human cytochrome c oxidase subunits migrated differently from their bovine counterparts. However, the position of subunits III and IV was the same in the human and in the bovine enzymes. The much higher mobility of human cytochrome c oxidase subunit II is explained by a greater hydrophobicity of this polypeptide than of that of the subunit II of the bovine enzyme.

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase.

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.

The effect of detergents on bovine cytochrome c oxidase: a kinetic approach.

(1) Investigation of the relationship between the detergent concentration and steady-state and pre-steady-state kinetics of cytochrome c oxidase proved to be a valid approach in the study of protein-detergent interaction. (2) Laurylmaltoside, sodium cholate and Triton X-100 influenced the kinetics of cytochrome c oxidase cooperatively at detergent concentrations near their critical micelle concentration. This mode of interaction reflects disaggregation of the oxidase as a result of cooperative binding of the detergent. (3) Addition of increasing concentrations of Tween-80 to the aggregated enzyme caused a more gradual decrease in aggregation of the oxidase, which did not result in a change in activity of the enzyme. This suggests that aggregation of cytochrome c oxidase occurs in a highly regular manner in which no catalytic sites are shielded off. (4) Oxidase aggregates present at detergent concentrations below the critical micelle concentration of laurylmaltoside and Triton X-100 showed considerable activity. Their kinetics were equal to those of the oxidase in Tween-80, suggesting that the protein molecules are aligned in a similar way in all oligomers. Aggregates present in low concentrations of sodium cholate showed turnover rates that were twice as low as those observed with other aggregates. (5) Solubilisation of the oxidase by sodium cholate or Triton X-100 resulted in almost complete inhibition of enzymic activity, whereas the association rate of ferrocytochrome c was almost equal to that found for monomeric oxidase in laurylmaltoside. These results are in agreement with a mixed-type inhibition.

Separation of enzymically active bovine cytochrome c oxidase monomers and dimers by high performance liquid chromatography

The aggregation state of two types of bovine heart cytochrome c oxidase preparations in the presence of laurylmaltoside was investigated by high performance liquid chromatography in two buffers of ionic strengths of 388 mM and 45 mM, respectively. At high ionic strength, it was found that the Fowler cytochrome c oxidase preparation was monomeric (Mr = 2 X 10(5)), while monomers and dimers (2 X aa3, Mr = 4 X 10(5)) could be isolated from the Yonetani preparation. Under these conditions there was no rapid equilibrium between the two forms. Covalent cytochrome c oxidase-cytochrome c complexes were largely dimeric, and addition of ascorbate and cytochrome c to the oxidase also promoted dimerization. At low ionic strength (I = 45 mM) in the presence of laurylmaltoside the oxidase and the covalent complex with cytochrome c were largely monomeric. In the steady-state oxidation of ferrous horse heart cytochrome c, the monomeric enzyme displayed biphasic kinetics at I = 45 mM. This suggests that the presence of high- and low-affinity reactions is an intrinsic property of the cytochrome c oxidase monomer.

Quantification of mitochondrial proteins in cultured cells by immuno-flow cytometry.

Immuno-flow cytometry was tested as a tool to estimate the cellular concentration of mitochondrial proteins in cultured cells, using cytochrome c oxidase as a model enzyme. Cells labelled with antibodies against cytochrome c oxidase, in which the amount of the enzyme was reduced by various extents, showed a linear relationship between the size of the signal obtained by immuno-flow cytometry and the amount of the enzyme. The determination by immuno-flow cytometry resulted in data comparable to the results obtained by immunoprecipitation and activity measurements. Since immuno-flow cytometry requires only limited numbers of cells, the method could especially be of value for diagnostic purposes. This is illustrated by the results obtained by comparing activity measurements and immuno-flow cytometry in the initial screening of cell lines derived from patients with deficiencies in the activity of cytochrome c oxidase.

Familial Mitochondrial Complex I Deficiency with an Abnormal Mitochondrial Encoded Protein

Six of seven male full sibs of Moroccan descent, consecutively indicated as A–G, had neonatal onset disease. Four (A, B, D, F) died, probably all from the same disorder. In four cases (D, E, F, G) neonatal onset lactic acidosis was documented. In cases E and G muscle biopsy was undertaken. A deficiency of NADH:Q1 oxidoreductse (complex I) was revealed in both. A liver biopsy in case G revealed the same abnormality. Patient histories and data on the mitochondrial function studies in cases E and G are given by Wijburg et al. (1989).

Cytochrome c Oxidase: Organ-Specific Isoenzymes and Deficiencies

Ever since the first report of Luft et al. (1962), considerable information has become available on inherited metabolic diseases originating from defects in mitochondria. Apart from defects in the import of substrates, in the carboxylic acid cycle, and in fatty acid oxidation, many dysfunctions have been described in the mitochondrial respiratory chain and ATP synthase (Morgan-Hughes, 1980). In the present study, we focused on defects of cytochrome c oxidase (E.C. 1.9.3.1), the terminal component of the mitochondrial respiratory chain.