Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

Abstract

The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a freeenergy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochrome c heme edge is within The determination of structures of bacterial and mitochondrial cytochrome c oxidases (CcO) 1 by x-ray crystallography (1-5) has opened the door to understanding the structural basis for its function in respiratory chain activity. One step of the respiratory chain involves the transfer of electrons from cytochrome bc 1 complex to CcO, both membrane-bound complexes, via the water-soluble protein cytochrome c (Cc). Efficient electron transfer requires both rapid complex formation and rapid product dissociation. Determination of the structure of the complex formed between CcO and Cc would reveal the interactions promoting this transient interaction as well as possible paths for intermolecular electron transfer. The complex structure, however, has yet to be determined, in part due to the difficulties in crystallizing membrane-bound proteins and possibly also due to the transient nature of the interaction. On the other hand, this redox system may be particularly amenable to computational search methods based on static crystallographic structures because of the fast kinetics of these interactions, which suggest that large conformational changes do not occur upon complex formation. In addition, computational techniques may shed light on long range electrostatic guidance between the two partners and on the dynamic nature of the interaction.The docking program DOT (6) provides a complete search of all orientations between two rigid molecules by systematically rotating and translating one molecule about the other, a procedure considered computationally unfeasible just a few years ago (7). At that time, the extent of the search was limited to, at most, energy calculations for a few million configurations, as in the programs TURNIP (8) and DOCK (9). Alternately, the representation of the moving molecule was greatly simplified to a few point charges (10, 11). DOT systematically samples configurations for two rigid molecules over all space and expresses the interaction energies as correlation functions, which are rapidly computed using fast Fourier transforms. The electrostatic potentials used by DOT are calculated by Poisson-Boltzmann methods (12-14), which take both solvent and ionic strength effects into account. Because DOT performs a complete search, both long range and short range interactions can be examined.Here, we describe the application of DOT to the interaction of bovine CcO with horse Cc. The calculated complex identifies the CcO Trp 104 (Trp 143 ) 2 side chain a...

Long-range electron transfer

“…Cu A , a binuclear site with bridging S(Cys) atoms, is the primary electron acceptor from cyt c. Studies with Ru-modified cyt c reveal rapid (6 ϫ 10 4 s Ϫ1 ) (84) electron injection from Fe(II) into Cu A at low driving force (⌬G°ϭ Ϫ0.03 eV) (85). Modeling suggests that cyt c binds to the enzyme at an acidic patch on subunit II (86,87). The cyt c heme is very near the Trp-104 (subunit II) indole ring, a residue that appears from mutagenesis experiments to be critical for rapid cyt c 3 Cu A ET (88)(89)(90)(91).…”

Rapid electrostatic evolution at the binding site for cytochrome c on cytochrome c oxidase in anthropoid primates

“…More recently, extensive mutational analysis (16,21,26) and simulated binding of horse CYC to bovine COX (15) further demonstrated the importance of electrostatic interactions between CYC and COX. The electrostatic interactions participate in long-range protein-protein association and short-range orientation, such as through salt bridge formation (15). Osheroff et al (23) examined by enzyme kinetic measurements the functional interaction between CYC and COX from different mammalian species.…”

“…However, against this background of slow rates of CYC and COX evolution, upsurges in COX and CYC amino acid replacement rates occurred during the emergence and evolution of the larger brained primates (members of Anthropoidea) (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Here, by using atomic resolution structural data on bovine COX (13) and horse CYC (14) and an experimentally verified model of the interaction between COX and CYC (15,16), we identify, among the Ͼ1,500 amino acid residues of COX, 57 residues that are likely to bind CYC during delivery of electrons to oxygen. We then demonstrate that the amino acid replacement rate acceleration in anthropoid lineages is especially pronounced in the subset of COX residues that can bind CYC.…”

“…When CYC and COX were from catarrhine sources, the enzyme kinetics were comparable with those measured when CYC and COX were from nonanthropoid mammals. The descriptions of the bovine COX (27) and horse CYC (14) crystal structures, investigations into the CYC-binding site of COX (15), and availability of a range of comparative COX and CYC sequence data from anthropoids and other vertebrate taxa now allow us to further examine evolutionary changes in electrostatic interactions between CYC and COX and to test whether CYC and COX coadaptively evolved in anthropoids.…”

“…COX residues that form the CYC binding site were extracted from these alignments by two criteria: that they lie on the surface of COX and that an atom of a surface COX residue side chain lies within 10 Å of any CYC atom when CYC is bound to COX (15). Surface residues were determined with the program GETAREA 1.1 (31) by using a radius for water of 1.4 Å. Polar interactions of up to 8.5 Å have been hypothesized in the docking model of CYC on COX (15). We have added 1.5 Å to allow for the possibility of longer range indirect interactions as well as minor conformational changes.…”