Volume Changes Associated with CO Photolysis from Fully Reduced Bovine Heart Cytochrome aa₃

Abstract

Volume changes associated with CO photodissociation from fully reduced cytochrome aa 3 have been determined using photoacoustic calorimetry (PAC). Analysis of PAC data reveals two intermediate steps in the photodissociation reaction with volume changes of +6.8 and +6.9 mL/mol occurring on time scales of <50 ns and ∼1.5 μs, respectively (T = 22 °C). The value of ΔV for the fast process can be attributed to conformational dynamics associated with photoinduced transfer of CO from the high-potential heme a 3 to the high-potential CuB site, while the slower phase reaction occurs on a time scale consistent with the thermal dissociation of CO from CuB. It is speculated that these volume changes may be linked to movement of Glu-242 as part of a proton shuttle mechanism in heme/copper oxidases.

Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase

“…In this case, the enthalpic changes for resolvable reactions steps, which occur subsequent to prompt heat release are described as DH i = Àq i . This is how Di Primo et al [40][41][42] reported DH values for CO photodetachment from cytochrome P450 cam and how Larsen and coworkers report their results [44][45][46][47][48][49][50][51][52]. Because we will compare our results to theirs, we follow this precedent in Section 4.…”

“…We will use this terminology in our initial analyses (see Tables 1 and 2, below) but will discontinue it when exploring effects of ionic strength on the thermodyamic parameters (Table 3, below), to facilitate comparison to results reported for the other heme proteins [40][41][42][44][45][46][47][48][49][50][51][52]. Thus to avoid confusion, we have explicitly defined our terminology for the enthalpic parameters here.…”

“…As a first approximation we assume they contribute equally to prompt heat release, and, that this prompt heat release is the major determinant of our observed q 1 , as Larsen et al [44] assumed for the analysis of the PA data from cytochrome bo 3 . While this assumption may be less well justified for FRCO cytochrome aa 3 [45], as the relative absorbance values at our excitation wavelength of 590 nm are simply estimated by Table 1 were obtained from the intercepts and DV values in Table 1 were given by the slopes of the lines shown here. The data was from samples in the lower ionic strength 50 mM HEPES at pH 7.4 with 0.2% n-dodecyl b-D maltoside buffer.…”

“…Larsen and coworkers have applied photothermal techniques to study CO photodetachment from heme-copper oxidases [44][45][46], model compounds [47][48][49], and smaller heme proteins [50][51][52]. They used photoacoustic measurements on both cytochrome bo 3 from E. coli [44,46] and bovine cytochrome c oxidase (cytochrome aa 3 ) [45]. The latter two proteins exhibit structural differences, however, both are terminal electron transport oxidases that pump protons across membranes in their respective environments.…”

“…Because these ligand transfers involve bond cleavages and bond formation, and are likely accompanied by protein conformational changes, these events give rise to rich, multicomponent PA signals. With a time window of approximately 50 ns to 5 ls, Larsen and Langley observed the enthalpic and volume changes associated with the CO transfer from heme a 3 to Cu B in fully reduced bovine cytochrome c oxidase (CcO) unresolved from the prompt heat release signal in <50 ns [45]. They also detected a volume change at approximately 1.5 ls in the eukaryotic system likely associated with the CO leaving its Cu B binding site.…”