Characterization of the 414 and 430 nm states of cytochrome a₃ in the resting form of cytochrome c oxidase
Papadopoulos, Pavlos George
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Incubation of resting cytochrome c̲ oxidase at low pH has two effects: 1) a blue shift of the Soret band to higher energy wavelengths with the occurrence of an isosbestic point, and 2) a fast to slow cyanide binding conversion. Variable Soret band positions reflect different mixtures of two forms of cytochrome a̲₃ with λ[sub max] values at 414 and 430 nm, representing protonated and deprotonated forms, respectively. It is shown in this thesis that there is no correlation between the 414/430 nm and the fast/slow cyanide forms under equilibrium conditions. A procedure for accurately determining the percentages of fast and slow cyanide conformers is presented, which allows the estimation of the expected final absorbance for the cyanide reaction at any given pH. Criteria are established to determine if any unreactive cytochrome a̲₃ centers exist following exposure to low pH. The extent of the blue shift in the Soret band (430 -> 414 nm conversion) is characteristic of the pH sensitivity of the enzyme. Treatments with a high concentration of n-dodecyl-β-D-maltoside (DM), high temperature, and protease are known to remove subunits from native cytochrome oxidase. These treatments are examined in order to test for any correlation between subunit composition and pH response. From empirical observations it is suggested that subunit Vila may be responsible for enhanced pH sensitivity. A model is proposed in which the 430 nm form can exist in two states: one, that is resistant to low pH and is referred to as [sub R], and a second that is pH sensitive and is referred to as [sub s]. According to the model, the high DM concentration results in an increase of the concentration of the [sub s] state. This cytochrome a̲₃ form is proposed to be related to the decrease in proton pumping efficiency that has been observed following DM treatment, by other investigators. The protease treatment induces optical changes that are very similar to those induced by an alkaline transition, including a red shift in the Soret band and an increase of the [sub s] concentration. Evidence is presented to critically examine whether the spectral changes are, in fact, due to protease, or some other component present in commercial preparations. Unlike the high DM treatment, proteolytic digestion (a-chymotrypsin or trypsin) does not enhance the low pH-induced sensitivity (no [sub R] -> [sub s] conversion). High temperature (30 - 40°C) also does not enhance the pH sensitivity of enzyme when incubated in 0.1 % (w/v) DM buffer. When the detergent concentration is increased to 2 % (w/v), high temperature incubation (5-6 hours) is proposed to cause a two state denaturation in cytochrome a̲ . Evidence is presented to indicate that longer incubation results in the denaturation of both cytochromes (a̲ + a̲₃).