Redox-Controlled Proton Gating in Bovine Cytochrome c Oxidase

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which proton pumping is coupled to the oxygen reduction reaction remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix. Here, with a novel hydrogen/deuterium exchange resonance Raman spectroscopy method, we have identified two critical elements of the proton pump: a proton loading site near the propionate groups of heme a, which is capable of transiently storing protons uploaded from the negative-side of the membrane prior to their release into the positive side of the membrane and a conformational gate that controls proton translocation in response to the change in the redox state of heme a. These findings form the basis for a postulated molecular model describing a detailed mechanism by which unidirectional proton translocation is coupled to electron transfer from heme a to heme a 3, associated with the oxygen chemistry occurring in the heme a 3 site, during enzymatic turnover.

2) Calculation of the changes in intensity in the resonance Raman difference spectra of the reduced enzyme ( Figure 2 in the main text).
As heme a and heme a 3 are not expected to have the same degree of the H/D exchange, their concentrations in the difference spectra may be expressed as follows: Eq. S1 where m and n are the fractional populations of heme a and a 3 , respectively, that underwent the D to H exchange within t. Therefore, if the exchange is complete, m (or n) will equal one and the difference spectrum will be featureless. In contrast, if there is no exchange, m (or n) will be zero and the difference spectrum will be the same as that of the reference difference spectrum.
3) Calculation of the changes in intensity in the resonance Raman difference spectra of the oxidized enzyme ( Figure 3 in the main text).
Thus, the heme a 3 modes are the same (fully exchanged) in both the reference (at time ~0) and in the time t spectrum so they cancel out and the remaining lines reflect the spectral changes in heme a only; the difference spectrum equation may then be written as: Analogous to the formula given by eq. 1, the standard H/D difference peaks of the heme a 2+ will appear in such a difference spectrum, if the H/D exchange at the heme a 2+ is hindered. In Figure S2, the difference spectrum at 180 minute (b) is compared with the standard H/D difference spectrum (a) for the reduced-CO form. The data show that the H/D exchange at the heme a 2+ is blocked in the reduced-CO form of the enzyme.
The investigations on the MV-SH¯ form were somewhat difficult as compared to those on the other forms, because of the stability of the form. The sulfide causes the reduction of heme a at one hand, and it binds to the heme a 3 3+ on the other to form the mixed valence (a 2+ + a 3 3+ -SH¯) configuration. The successful preparation and the stability of the form depend on the experimental conditions, such as the concentration of the sulfide [1]. In this study, we added 1 mM sodium sulfide aerobically to the CcO Ox D sample, and incubated the sample (for ~15 minutes) until the optical absorption showed the complete formation of the MV-SH¯ form. The resulted MV-SH¯ sample was diluted (by 1:9 ratio) into an H 2 O buffer, which included 1 mM sodium sulfide. Five minutes after the dilution, resonance Raman spectra were measured repeatedly every 10 minutes. In Figure S7, the resonance Raman data are expressed in a form In the difference spectra, the presence of the H/D difference peaks is a measure for the absence of the H/D exchange at the heme a 2+ . The resonance Raman data showed that the A and D propionate peaks seen in the standard difference spectrum ( Figure   S7, spectrum (c)) remained after exposure to the protonated buffer from 5-25 minutes.
Resonance Raman data taken at time points later than those shown here were not reliable, because the heme a in the sample gradually became oxidized (data not shown). Although the time frame of the investigation was limited, the present results show a persistent lack of H/D exchange at heme a 2+ in the MV-SH¯ form.
It is well established that the P M form of bCcO is generated, when resting bCcO is exposed to a mixture of CO and O 2 gasses; CO is acting as a reducing agent for heme a 3 , while the O 2 molecule is catalyzed at heme a 3 2+ , leading to the a 3 4+ =O 2heme [2]. We incubated the CcO Ox D sample in a 1:1 mixture of CO and O 2 for 15 minutes to make a P M preparation (termed CcO P M D ). The CcO P M D sample was then anaerobically transferred into a Raman cell filled with 100 % CO. In the Raman cell, the sample was diluted anaerobically with the H 2 O buffer, We continuously recorded the visible-absorption spectrum for each sample from immediately after the dilution to before the reduction by dithionite, and made sure that the sample was always in the P M form during the incubation period. Although the exact extinction coefficients have not reported for the P M form made by the present method, its Soret-visible absorption spectrum was given in comparison with other forms of bCcO [3]. On the basis of such spectral comparisons, together with known extinction coefficients of bCcO, we estimated an extinction coefficient of P M form to be 21 mM -1 cm -1 (ε 604-630 ), and also determined that of the oxidized form for the same wavelength interval (604 nm to 630 nm) to be 13.6 mM -1 cm -1 .
Using these extinction coefficient values, the population of the P M form during the incubation period was determined to be >87 %.
The dilution/reduction sequence described here was the same as that we used for testing the H/D exchange in the resting state, although the starting material before the dilution was CcO P M D instead of CcO Ox D , while the final product was in the reduced-CO state instead of the fully-reduced state. Accordingly, analogous to the formulation of Eq. S2, we can draw the following scheme for analyzing the resonance Raman spectra from the [ indeed showed a time dependent increase of the amplitude ( Figure S8), which from which the rate of exchange was determined to be 3 (± 1) x 10 -1 min -1 .

5) H/D exchange in the pulsed enzyme.
The resting form of CcO is converted to a slightly different form, called the "pulsed form", immediately following turnover of the enzyme and that the pulsed form of the oxidized enzyme exhibits a higher activity [4].  Figure S6 (Spectrum c and b, respectively). The data show that the two difference spectra are almost identical, demonstrating that the H/D exchange in the pulsed enzyme is the same as that in the resting enzyme.
6) The role of water channels in proton translocation.
The H-channel is composed of a series successive water accessible cavities leading from the n-side surface of the membrane to the vicinity of the heme a [5,6]. Combining the crystal structure data, in which the cavity sizes in the oxidized state of the enzyme are reduced [6], with site-directed mutational studies [5], a model was proposed in which water passage through the cavities is allowed in the reduced state of the enzyme and prohibited in the oxidized state [7]. It was postulated that protons are then translocated from the water pockets along heme a to D51 on the p-side of the membrane controlled by the heme a oxidation state and the heme a 3 ligand binding states. In that model, the gate was ascribed to the presence of pockets that could translocate water molecules in the reduced structure but not in the oxidized structure.
The gate observed in this study is, however, not assignable to the gate in the water pockets, as the H/D exchange data demonstrate that the gate is closed in the reduced state and open in the