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The molecule at left is an important proton pump in the mitochondrial electron transport chain (ETC).
The electron transport chain is a chain of enzymes that undergo a series of reduction-oxidation (redox) reactions. As each complex in the chain goes through a series of redox reactions, the enzymes release protons into the intermembrane space. This proton release increases the electrochemical proton gradient, which is the driving force for ATP synthesis by ATP synthase (Wikstrom and Sharma, 2018). See Figure 1 for a systematic view of this process. In this study we display and describe the terminal enzyme in the electron transport chain, Cytochrome C Oxidase (CcO), commonly known as Complex IV, of the bovine heart. CcO is responsible for catalyzing the last step of cell respiration in all mitochondria (Wikstrom and Sharma, 2018). The enzyme accepts an electron from Cytochrome C (CytC) to reduce O2 into water. This electron pathway works synergistically with pumping protons into the intermembrane space.:
4 Cyt Creduced + 4 H+ + O2 + 4H+matrix --------> 4 Cyt Coxidized + 2H2O + 4H+intermembrane space
The bovine heart CcO contains 13 different subunits, illustrated as
multi-subunited polymer. To the left we demonstrate CcO with the 13 subunits differentiated by color. The 3 largest subunits are coded by mitochondrial DNA while the other 10 are nuclear coded. The bulk of this enzyme lies in the intermembrane of mitochondria. The top of the enzyme is positively charged due to the proton gradient while the bottom in negatively charged (Yoshikawa et al, 2012).
II. O2 Reduction
The electron accepted from CytC goes through various molecules to reach the O2 reduction site. Outlined below is the electron pathway through the enzyme, the O2 reduction site and the proton pathway used in the reduction of O2 to H2O.
An electron is accepted from CytC by Copper A (CuA) in CcO. This site is a binuclear center. The electron donation reduces the copper site to an unfavorable state. This promotes CuA to return to its oxidative state releasing an electron to the low-spin heme, Heme A. Heme A then transfers the electron to the O2 reduction site. In this site, the electron is first accepted by the high-spin heme, Heme A3. Then, this electron is transferred to Copper B (CuB).
The dioxygen reduction site consits of Heme A3, CuB, and Tyr244. Due to the high unfavorable reduction of just one oxygen atom, both metal sites must be in the reduced state to accept O2. Initially, O2 binds to CuB transiently through the O2 transfer pathway. Then the O2 moves and binds to Heme A3, receiving one electron to form O2-. A fixed water molecule is introduced, triggering a non-sequential 3-electron reduction of O2-. This reduction involves HemeA3, O2-, CuB and the OH group in Tyr244.
Through the D- and K- channels, protons are transferred to the O2 reduction site. These protons bind to intermediates of O2 reduction to form water. The D- and K-channels are named after amino acid residues Asp91 and Lys319 respectively.
III. Proton-pumping Process
As the electron from CytC initiates a redox series through the enzyme, this reaction is coupled with the proton pumping force. A study done by Tsukihara et al. 2003, found that the low-spin heme (Heme A) is the driving force for the proton-pumping process. The reduction of Heme A, causes a conformational change in the enzyme forcing Asp51 to be exposed to the intermembrane space. Due to this, Asp51 loses a proton, which adds to the proton electrochemical gradient for ATP synthesis. Asp51 is then re-protonated through a hydrogen bonded network. Below are few conformational changes that occur in the proton-pumping pathway.
Shown to the left is a conformational change in both the oxidized state and the reduced of CcO. When Heme A is reduced, a conformation change is seen in Asp51. In the oxidized state Asp51 is protonated and buried inside the protein. During Heme A reduction the removal of the net positive charge exposes Asp51 to the intermembrane surface. This exposure causes a pKa change in Asp51’s environment, which in turn releases a proton into the intermembrane space. Upon oxidation of Heme A, Asp51 moves back into the protein.
As Asp51 is deprotonated, the net positive charge on Heme A during oxidation, promotes proton transfer from Arg38 toward Asp51 through a hydrogen-bonded network. The hydrogen bonded network consists of the Asp51, Ser441, Tyr440, Tyr371, Arg38 and two fixed water molecules (Tsukihara et al., 2003).
The re-protonation of Arg38 occurs during the reduction of Heme A. When Heme A is reduced, the hydrofarnesylethyl group rotates 120°. This rotation causes a conformational change that creates a new cavity in the water channel. The increase in capacity allows for more water molecules to be taken up from the matrix space. The deprotonated Arg38 extracts a proton from a water molecule in the water channel. In the oxidized state, Arg38 is predominately protonated due to the encounter with water molecules in the water channel. The hydrogen bonded network and the water channel work in tandem to form the H-pathway (Yoshikawa et al., 2012).
All these steps put together create a method for the enzyme to release a proton by exposing Asp51 to the intermembrane space. Then re-protonating Asp51 by allowing a proton transfer from the matrix to Asp51 through a hydrogen bonded network.
The reduction of O2 and the proton-pumping process are a coupled reaction in Cytochrome C Oxidase. This coupled reaction leads to a 1-to-1 ration of electrons used to protons pumped. Together CcO increases the proton motive force for ATP synthesis and uses O2 as the final electron acceptor in the electron transport chain.
Tsukihara, T et al. 2003. The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process. PNAS 100:15304-15309.
Wilstrom M. and V. Sharma. 2018. Proton-pumping by cytochrome c oxidase- A 40-year anniversary. Elsevier 1859: 692-698.
Yoshikawa, S. et al. 2012. Structural studies on bovine heart cytochrome c oxidase. Elsevier 1817:579-589.