Stuchebrukhov research group           Department of Chemistry           University of California at Davis

Dependence of pKa's on Conformational Changes of Glu242 Residue in Bovine CcO

Popovic & Stuchebrukhov, PPS, 5, 611 (2006)

The objectives of this work is to answer the following questions: How do the presence or absence of a proton on the Glu242 and/or His291 site affect the pKa values of the other partner? How do conformational changes of theGlu242 side chain affect the pKa values of the two residues in different redox states of the enzyme? What is the coupling between the redox states of heme a or CuB center and the protonation states of Glu242 and His291 residues? How do the redox state and redox potential of heme a depend on the protonation state ofGlu242, and vice versa?

The two conformations of Glu242 side chain                    Water chain between Glu242 and PRD of heme a3

The coordinates of the downward (red) conformation are taken from the X-ray structure of bovine heart cytochrome c oxidase; in this state Glu242 is in contact with the D-channel.

The upper (blue) conformation is obtained by an ab initio computation of a system consisting of the Glu242 side chain,propionate group (representing PRD of heme a3) and three water molecules in between, forming a stable H-bonded chain. The upper conformation is presumably the proton releasing conformation of Glu242 residue.

The schematics of the proposed proton-pumping mechanism of CcO. The sequence of transitions during one pumping cycle of the enzyme is shown. The redox state of CuA, heme a, heme a3 and CuB center, and the protonation state of OH/H2O ligand to CuB, His291 and Glu242 sites are displayed. Red arrows represent the ET, while the proton translocations are designated by blue arrows.

The mutual dependence of the pKa values of His291 and Glu242 on their protonation states and conformation of Glu242 side chain, for the dielectric medium of εprot = 4 and εcavity = 20. The sequences of numbers correspond to OORO,ORRO and OORR redox states, respectively. The pKa value of His291 depends on the protonation and conformation state of Glu242: A) Glu protonated and downwards, B) Glu protonated and upwards, C) Glu deprotonated and downwards, D) Glu deprotonated and upwards. The pKa of Glu242 depends on the orientation of its side chain (down/up) and the protonation state of His291: E) His protonated, F) His deprotonated.

pKa of Glu242 vs. Redox Potential of Heme a

The coupling of the redox state of heme a and protonation state of Glu242, calculated at pH = 7. The heme a is represented by the central Fe atom and by a line defining the porphyrin ring. It is only shown a formal oxidation state of Fe atom.


Recent calculations from our group indicate that His291, a CuB-ligand, may play the role of the proton loading site. In this work, we explore the coupling of the conformational changes of Glu242 residue (the main proton donor of both chemical and pump protons) to its pKa, and the pKa of His291. The computations are done for several redox states of metal centers, different protonation states of Glu242 and His291, and two defined conformations of the Glu242 side chain (a proton input and output conformation). Thus, in addition to equilibrium redox/protonation states of the catalytic cycle, the transient and intermediate states are also examined. Different dielectric models are employed to investigate the robustness of the results, and their viability in the light of the proposed proton pumping mechanism of CcO.

The main results are in agreement with the experimental measurements and support the proposed pumping mechanism. Additionally, the present calculations indicate a possibility of gating through conformational changes of Glu242; namely, in the pumping step, we find that Glu242 needs to be reprotonated before His291 can eject a proton to the P-site of membrane. As a result, the reprotonation of Glu can control proton release from the proton loading site.

We have also explored the mutual coupling between the protonation state and pKa of Glu242 and the redox state and potential of heme a. We find that the protonation state of Glu is independent of the redox state of heme a, though its pKa value is sensitive to the redox changes of the system. On the other hand, the reduction of heme a is only possible if the Glu242 site is protonated. The strength of the coupling between the two groups is about 170 meV at neutral pH, which is in good agreement with the experiments.