Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein.

Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.

Synthesis of cytochrome c oxidase models bearing a Tyr244 mimic.

A close structural analogue of the metal-free cytochrome c oxidase active site has been synthesized. This model has a proximal imidazole tail and three distal imidazole pickets attached to a porphyrin. One distal imidazole is cross-linked to a phenol, mimicking Tyr(244). The strategy behind the successful synthesis of this regioisomerically pure model involved discovering the best sequence to introduce the phenol-substituted imidazole and employing a fluorinated substituent.

Binding of a synthetic targeting peptide to a mitochondrial channel protein.

Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) from Neurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochrome c in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochrome c bind to protein "arms" which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.

Cytochrome c oxidase: a new conformational model

Cytochrome c oxidase and zinc cytochrome c were cross-linked to form a 1:1 enzyme-substrate convalent complex. The reaction was catalyzed by 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) and cytochrome c, the substrate, became attached to the binding site located at submit II of the enzyme. This covalent complex could still be reduced by unbound ferrocytochrome c, suggesting the existence of more than one substrate binding site [1]. Previously [2] we reported that the reduction of heme a triggered a major conformational change within cytochrome c oxidase. Here we propose a model which incorporates these two findings with other established features of cytochrome c oxidase. The most prominent features of this model are (i) Electrons may enter the enzyme via both heme and CUa. Thus cytochrome oxidas may be regarded as having two substrate binding sites, one near heme a and the other near CUa. (ii) At any instant however, only one site is normally available for the binding of substrate. (iii) Subsequent to binding, a substrate molecule transfers an electron to the redox center at the site; reduction of the redox centre triggers a conformational transition which abolishes that binding site and simultaneously leads to the formation of an adjacent substrate binding site near the other redox centre. (iv) The two sites possess approximately equivalent affinity for substrate(AU)