Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

Abstract

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (-)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O(2) consumption can be used to compare the rate and functional pK(a) of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (-) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake.

Figure 1

The D pathway of the aa₃-type CcO of Rhodobacter sphaeroides. Subunit I is shadowed in brown, subunit III in green. The two phospholipids bound within the cleft of subunit III are in purple. The structure shown is PDB 1M56 [17] since this is the only structure to date of wild-type R. sphaeroides CcO that resolves subunit III to high resolution. Seven waters of the D pathway are shown in cyan.

Figure 2

pH dependence of steady-state activity by wild-type CcO and CcO III (−) forms with different initial proton acceptors for the D pathway (see Table 1). The data indicate the distribution of functional pK_as of proton uptake and the extension of the pH range of enzyme activity afforded by manipulation of the proton uptake step. Each data set, taken from Varanasi & Hosler [27], is normalized by setting the highest measured activity as 100%; fits are to the Henderson-Hasselbalch function. The functional pK_a values are presented in Table 1.

Figure 3

pH dependence of the catalytic lifespan of WT III (−), with Asp-132 as the initial proton acceptor of the D pathway, and H26D-D132A III (−), with Asp-26 as the initial acceptor. Catalytic lifespans were measured for the purified CcO forms in dodecyl maltoside solution supplemented with exogenous soybean phospholipid as detailed in Mills and Hosler [35] using the buffer systems of Varanasi and Hosler [27]. The data for WT III (−) is fit to a single exponential decay function using GraphPad Prism.

Figure 4

pH dependence of the catalytic lifespan of CcO III (−) forms with Asp-132, Asp-139, Asp-132 plus Cys 139 or Asp-132 plus Asp-139 as the initial proton acceptors of the D pathway, compared to normal CcO containing subunit III (WT III (+). The green line represents the minimum catalytic lifespan of WT III (+) across this pH range; the long lifespan of WT III (+) is difficult to measure with accuracy. Catalytic lifespan measurements and exponential fits were performed as in Figure 3.

Figure 5

The exponential relationship of catalytic lifespan to the rate of steady-state proton uptake for CcO III (−) forms with Asp-132 (WT III (−) or Asp-132 plus Asp-139 (N139D III (−) as initial proton acceptors of the D pathway. The rate of steady-state proton uptake of substrate protons is the turnover number, expressed as H⁺ consumption (H⁺ s⁻¹). The Y axis on the left shows the CC₅₀ values measured for WT III (−) while the ten-fold higher CC₅₀ values of the Y axis on the right are those measured for N139D III (−). The data are fit to a single exponential growth functions using GraphPad Prism.