Abstract
Cellular respiration is a fundamental process required for energy production in many organisms. The terminal electron transfer complex in mitochondrial and many bacterial respiratory chains is cytochrome c oxidase (CcO). This converts the energy released in the cytochrome c/oxygen redox reaction into a transmembrane proton electrochemical gradient that is used subsequently to power ATP synthesis. Despite detailed knowledge of electron and proton transfer paths, a central question remains as to whether the coupling between electron and proton transfer in mammalian mitochondrial forms of CcO is mechanistically equivalent to its bacterial counterparts. Here, we focus on the conserved span between H376 and G384 of transmembrane helix (TMH) X of subunit I. This conformationally-dynamic section has been suggested to link the redox activity with the putative H pathway of proton transfer in mammalian CcO. The two helix X mutants, Val380Met (V380M) and Gly384Asp (G384D), generated in the genetically-tractable yeast CcO, resulted in a respiratory-deficient phenotype caused by the inhibition of intra-protein electron transfer and CcO turnover. Molecular aspects of these variants were studied by long timescale atomistic molecular dynamics simulations performed on wild-type and mutant bovine and yeast CcOs. We identified redox- and mutation-state dependent conformational changes in this span of TMH X of bovine and yeast CcOs which strongly suggests that this dynamic module plays a key role in optimizing intra-protein electron transfers.
Highlights
All forms of cytochrome c oxidase (CcO) reduce molecular oxygen (O2) to water with four electrons provided by cytochrome c and four protons from the mitochondrial matrix or bacterial cytoplasmic aqueous phase
V380M and G384D are located on the helix X face opposite to S382 and extend towards the binuclear center (BNC) (Figure 1B), and are quite separate from all three proposed proton channels (Supplementary Figure S1)
We suggest that in addition to the conformational effects discussed above for G384D mutant, lowered electron affinity of heme a3 contributes to the observed enzymatic inactivity, and is in agreement with our experimental data that show that electron transfer from heme a to heme a3/CuB is blocked
Summary
All forms of cytochrome c oxidase (CcO) reduce molecular oxygen (O2) to water with four electrons provided by cytochrome c and four protons from the mitochondrial matrix or bacterial cytoplasmic aqueous phase. These charge transfers from the two opposite sides of the membrane result in membrane polarization, which is further enhanced with an energetically uphill transfer of four more protons across the membrane (Wikström, 1977) (Figure 1A). The resulting proton concentration difference and charge imbalance across the membrane (the protonmotive force) drives the synthesis of ATP. The redox chemistry at the BNC drives the proton pump of CcO.
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