Characterisation of 'fast' and 'slow' forms of bovine heart cytochrome-c oxidase
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A J Moody1, C E Cooper, P R Rich
1Glynn Research Institute, Bodmin, U.K.
Insights
Researchers studied bovine heart cytochrome-c oxidase, identifying distinct "fast" and "slow" forms. The "slow" form, resembling the enzyme's resting state, can be induced by incubation, suggesting a ligand interaction at the binuclear center.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein structure-function relationships
Background:
- Cytochrome-c oxidase (COX) is a crucial enzyme in cellular respiration.
- COX exists in different conformational states, impacting its activity and ligand binding.
- Understanding these states is key to elucidating COX function and regulation.
Purpose of the Study:
- To characterize the distinct "fast" and "slow" forms of bovine heart cytochrome-c oxidase.
- To investigate the interconversion between these forms and the role of ligands.
- To correlate these forms with the known "pulsed" and "resting" states of COX.
Main Methods:
- Enzyme preparation using a modified Kuboyama et al. method.
- Spectroscopic analysis (Soret absorption, EPR) to characterize enzyme states.
- Kinetic assays for cyanide and formate binding.
- Enzyme incubation under varying pH and ligand conditions (formate, chloride, bromide).
Main Results:
- A "fast" COX form was prepared, characterized by rapid cyanide binding and a high-spin heme a3 signal.
- Incubation at pH 6.5 converted the "fast" form to a "slow" (resting) form with altered kinetics and spectral properties.
- Formate, chloride, and bromide binding modulated COX activity, with formate mimicking a ligand in the "slow" form's binuclear center.
- Evidence suggests an equilibrium between high-spin and low-spin heme a3 in "fast" COX subpopulations.
Conclusions:
- The study establishes distinct "fast" and "slow" forms of bovine heart COX.
- "Slow" form characteristics suggest the presence of a binuclear center ligand, potentially mimicked by formate.
- The findings support the equivalence of "fast"/"slow" forms to "pulsed"/"resting" COX states, offering insights into enzyme regulation.
Abstract:
We have prepared cytochrome-c oxidase from bovine heart (using a modification of the method of Kuboyama et al. (1972) J. Biol. Chem. 247, 6375-6383) which binds cyanide rapidly, shows no kinetic distinction between the two haems on reduction by dithionite, has a Soret absorption maximum above 424 nm, and has a negligible 'g' = 12' EPR signal. On incubation at pH 6.5 this 'fast' oxidase reverts to the 'slow' ('resting') form characterised by slow cyanide binding, slow reduction of haem a3 by dithionite, a blue-shifted Soret maximum and a large 'g' = 12' signal. Incubation of 'fast' oxidase with formate produces a form of the enzyme with properties almost identical to those of 'slow' oxidase. The kinetics of formate binding to 'fast' oxidase are found to be biphasic, revealing the presence of at least two 'fast' subpopulations in our preparations. Evidence is presented that there is an equilibrium mixture of high-spin and low-spin forms of haem a3 in both 'fast' subpopulations at room temperature. Incubation of 'fast' oxidase with chloride or bromide at pH 6.5 produces forms of oxidase with much lower rates of cyanide binding. Our working hypothesis is that formate mimics a binuclear centre ligand which is present in the 'slow' form of cytochrome oxidase. Although we show that chloride and bromide can also be ligands of the binuclear centre, possibly onto CuB, we can rule out either of these being the ligand present in the 'slow' enzyme. We will argue that the 'fast' and 'slow' forms of oxidase are equivalent to the 'pulsed' and 'resting' forms of oxidase, respectively.