Control of Electron Transfer to the Binuclear Center in Cu-Heme Oxidases

Abstract

Figure 1 depicts the structure of the active site of cytochrome c oxidase, including cytochrome a and the (oxygen binding) binuclear center cytochrome a ₃-Cu_B as obtained from the crystallographic data on the beef heart enzyme [1]. One of the transmembrane helices of subunit I, helix X, provides two His as ligands of cytochrome a (His378) and cytochrome a ₃ (His376). Laser photolysis experiments starting from the CO adduct of the fully reduced or mixed-valence enzyme from beef heart yield rate constants for internal ET ranging from 10⁴ to 3 × 10⁵ s^-1 [2–4]. These very rapid rates are consistent with the short distance (13 Å) separating the two metals which are connected via a possible covalent pathway, involving 16 bonds [5]. On the other hand, it has been reported in the literature that the apparent rate of formation of reduced cytochrome a ₃, either in the presence or in the absence of CO, is considerably slower (0.1 to > 30 s^-1 depending on experimental conditions [6–8]). In spite of some complexity related to the state of the enzyme (whether “resting” or “pulsed”), this apparent rate constant seems to correlate with the turnover number of the enzyme; thus Malatesta et al. [8] concluded that internal electron transfer to the oxidized binuclear center is the rate-limiting step in turnover. More recently Verkhovsky et al. [9] confirmed that the rate of formation of reduced cytochrome a ₃ is slow (ms); however they assumed that even in the oxidized enzyme, internal electron transfer is very fast (μs), but the redox equilibrium between cytochrome a and cytochrome a ₃ favors the former, and proton diffusion and/or binding to the reduced binuclear site is the rate limiting step. In summary, there is substantial agreement about the basic experimental observation, i.e. that starting from oxidized cytochrome c oxidase the rate of formation of reduced cytochrome a ₃ is in the ms time range; nevertheless two alternative mechanisms have been proposed.