Computational study of the activated O _H state in the catalytic mechanism of cytochrome c oxidase

Abstract

Complex IV in the respiratory chain of mitochondria and bacteria catalyzes reduction of molecular oxygen to water, and conserves much of the liberated free energy as an electrochemical proton gradient, which is used for the synthesis of ATP. Photochemical electron injection experiments have shown that reduction of the ferric/cupric state of the enzyme's binuclear heme a 3 /Cu B center is coupled to proton pumping across the membrane, but only if oxidation of the reduced enzyme by O 2 immediately precedes electron injection. In contrast, reduction of the binuclear center in the "as-isolated" ferric/cupric enzyme is sluggish and without linkage to proton translocation. During turnover, the binuclear center apparently shuttles via a metastable but activated ferric/cupric state (O H ), which may decay into a more stable catalytically incompetent form (O) in the absence of electron donors. The structural basis for the difference between these two states has remained elusive, and is addressed here using computational methodology. The results support the notion that Cu B [II] is either three-coordinated in the O H state or shares an OH − ligand with heme a 3 in a strained μ-hydroxo structure. Relaxation to state O is initiated by hydration of the binuclear site. The redox potential of Cu B is expected, and found by density functional theory calculations, to be substantially higher in the O H state than in state O. Our calculations also suggest that the neutral radical form of the cross-linked tyrosine in the binuclear site may be more significant in the catalytic cycle than suspected so far.oxygen reduction | electron transfer

Proton-coupled electron transfer and the role of water molecules in proton pumping by cytochrome c oxidase

“…To further test whether the presence of a H-bond acceptor in the BNC compromises the water-gated mechanism, we performed fairly long (∼70-ns) MD simulations in yet another version of the F states (F H ' and F H,R ). These states are similar to F' and F R , except that the water ligand of Cu B has been removed (26), which leaves the ferryl iron-oxo group as a strong hydrogen bond acceptor in the BNC. The data presented in Table 2 show a similar trend as for the other intermediates.…”

“…Therefore, a water molecule rather than a hydroxyl ion most likely ligates the Cu B ion in this state (Table 1; see also ref. 26). This difference is found to be very important and is assessed below.…”

“…The protonation states of BNC ligands were decided on the basis of available experimental data (3) and previous density functional theory (DFT) calculations performed on cluster systems (3,21,26). It is known based on spectroscopic measurements that all P and F variants have a ferryl heme a 3 (3,14) except for the postulated state F R , which has ferric heme a 3 ligated by a hydroxyl ion (26).…”

“…It is known based on spectroscopic measurements that all P and F variants have a ferryl heme a 3 (3,14) except for the postulated state F R , which has ferric heme a 3 ligated by a hydroxyl ion (26). Cu B , on the other hand, is known to be ligated either by an OH in all P, or by a water molecule in all F states (14), with the exception of state F R (ref.…”

“…The charges and parameters of standard amino acid residues were obtained from the Chemistry at Harvard Macromolecular Mechanics (CHARMM) force field (40), and for metal centers, charges and parameters were obtained from previous studies (21,26). After construction of the protein system, it was immersed in a lipid bilayer comprising cardiolipin (CL), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) molecules.…”