Probing the specifics of substrate binding for cytochrome c oxidase: a computer assisted approach.

A deficiency of cytochrome c oxidase (COX) is associated with a number of diseases but details of the enzyme's mechanism of action especially the interaction with its substrate, ferrocytochrome c, remain unclear. It is known that the transfer of electrons from ferrocytochrome c to COX is facilitated by the formation of enzyme-substrate (ES) complexes which are stabilized by intermolecular salt bridges, however the identity of residues participating in the salt bridges remains obscure. Using the published structures of the two proteins, computer simulations were employed to model their interactions and to attempt to identify residues that participate in intermolecular salt bridges. The simulation process was guided in the main by cross-linking studies which proposed that Lys-13 of cytochrome c is paired with Asp-158 of COX. The initial enzyme-substrate complex, created by computer assisted manipulation of the two structures exhibited three salt bridges; following the application of energy minimization procedures, the number of salt bridges increased to seven and there were twenty-four intermolecular hydrogen bonds. The salt bridges emanated from: Glu-119 and Asp-221 of subunit I; Glu-114, Asp-115 and Asp-158 of subunit II and Asp-73 and Glu-78 of subunit VIb. These were paired with Lys-87, 8, 25, 27, 13, 22 and 100 respectively of cytochrome c. These results suggest that subunits I, II and VIb play direct roles in substrate binding. The results also suggest that hydrogen bonds contribute significantly to the stability of the ES-complex.

Early electron transfer in cytochrome c oxidase occurs by a chymotrypsin type relay.

Evidence suggests that when ferrocytochrome c (the substrate) reduces cytochrome c oxidase (COX), electrons from the former enter the latter via Trp-104. What is still to be determined is the method by which electrons are transferred from ferrocytochrome c to Trp-104 and the method by which electrons arriving at Trp-104 are moved on to Cu(A), the first of the enzyme's four redox centres to be reduced. To shed light on this process, we used the computer to create and analyse an enzyme-substrate complex formed from the published structure of the two proteins. It was found that the front haem edge of ferrocytochrome c was in close proximity to Trp-104 of COX and that inclusive of Trp-104, only nine amino acid residues from COX lie along a broad channel stretching from Trp-104 to the enzyme's Cu(A) centre. Six of the nine residues, Trp-104, Tyr-105, His-102 Trp-106, Asp-158 and Glu-198, had the ideal chemical properties and were properly aligned to facilitate electron transfer. Here we propose that the reduction of Trp-104 and the subsequent reduction of Cu(A) occur by a hydride/hydrogen ion relay system similar to that seen at the active site of chymotrypsin.

Cytochrome c/cytochrome c oxidase interaction. Direct structural evidence for conformational changes during enzyme turnover.

We investigated the interaction between cytochrome c oxidase and its substrate cytochrome c by catalyzing the covalent linkage of the two proteins to yield 1 : 1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the 'traditional' oxidized complex formed between oxidized cytochrome c and the oxidized enzyme we prepared complexes under steady-state reducing conditions. Whereas for the 'oxidized' complex cytochrome c became bound exclusively to subunit II of the enzyme, for the 'steady-state' complex cytochrome c became bound to subunit II and two low molecular mass subunits, most likely VIb and IV. For both complexes we investigated: (a) the ability of the covalently bound cytochrome c to relay electrons into the enzyme, and (b) the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Steady-state spectral analysis (400-630 nm) combined with stopped-flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. In the case of the latter, the half life for the ascorbate reduction of the bound cytochrome c and that for the subsequent transfer of electrons to haem a were both < 5 ms. In contrast the covalent complexes, when reduced, were found to be totally unreactive towards oxidized cytochrome c oxidase confirming that the previously observed reduction of haem a within the complexes occurred via intramolecular rather than intermolecular electron transfer. Additionally, stopped-flow analysis at 550 nm showed that haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochrome c: The second order rate constant for the traditional complex was 0.55x10(6) m(-1) x s(-1) while that for the steady-state was 0.27x10(6) m(-1) x s(-1). These values were approximately 25-50% of those observed for 1 : 1 electrostatic complexes of similar concentrations. These results combined with those of the ascorbate and the electrophoresis studies suggest that electrons are able to enter cytochrome c oxidase via two independent pathways. We propose that during enzyme turnover the enzyme cycles between two conformers, one with a substrate binding site at subunit II and the other along the interface of subunits II, IV and VIb. Structural analysis suggests that Glu112, Glu113, Glu114 and Asp125 of subunit IV and Glu40, Glu54, Glu78, Asp35, Asp49, Asp73 and Asp74 of subunit VIb are residues that might possibly be involved.

Investigating the enzyme-substrate interactions of cytochrome c oxidase: a computer-assisted approach [abstract]

Although the enzyme cytochrome c oxidase (COX) is critical to respiration and has been studied extensively, the interactions between this enzyme and its substrate cytochrome c are still not very well understood. We employed a computer assisted approach to study these interactions. We used the Swiss-pdb v 2.5 computer programme, which measures the percentage accessibility of residues and the online server ANOLEA, which calculates the non-local energy of residues in a polypeptide chain, to analyze the respective molecular structures of cytochrome c and COX. The accessibility studies showed that, compared to the oxidized, the reduced form of cytochrome c normally had the greater proportion of more highly accessible residues: for reduced cytochrome c, 7 of the 8 residues exhibiting 55 percent accessibility were lysines. Interestingly enough, lysine 13, shown by other studies to be important for substrate binding, was not significantly accessible. For COX, neither asparate 158 nor glutamate 198 residues, reported to be important for substrate binding and catalysis, were significantly accessible either. The energy studies showed that whereas oxidized cytochrome c was a stable structure of low energy, approximatley 81 percent of the protein was reduced. For COX, a few small regions, including 4 residues in the vicinity of CuA, which functions as the enzyme's electron entry port, were of high energy. Since lysine 13, aspartate 158 and glutamate 198 are known to play important roles in enzyme-substrate interactions, it must be that these residues become more accessible when the two proteins interact. The results of the accessibility studies therefore appear to suggest that COX employs a mix of induced-fit and strain mechanisms when it binds substrate. On the basis of the energy studies, we conclude that the structure of reduced cytochrome c (the substrate) resembles a high energy transition-state intermediate and that when this protein binds and reduces oxidized COX, the structures of both proteins are stabilized. (AU)

Cytochrome c oxidase: structural evidence for conformational switching between two substrate binding sites [abstract]

Cytochrome c oxidase, the final member of the electron transport chain is critical to aerobic respiration and the absence, deficiency or malnutrition of this enzyme causes a number of myopathies and other diseases, some of which are fatal. In spite of its importance, the enzyme is still not well understood and whether one or two binding sites for its substrate cytochrome c remains unresolved. In an attempt to answer this question, we prepared 1:1 covalent enzyme-substrate complexes under conditions of low ionic strength. In addition to the `traditional' complex formed between oxidized cytochrome c and the oxidized enzyme, we prepared a new complex under `steady-state' reducing conditions. For both complexes, we investigated the ability of the covalently bound enzyme to catalyze the oxidation of unbound (exogenous) ferrocytochrome c. Whereas for the `traditional' oxidized complex, cytochrome c became bound exclusively to subunit II of the enzyme, for the `steady-state'complex, cytochrome c became bound to subunits II, VIa and VIb. Steady-state spectral analysis (400-630nm), combined with Stopped-Flow studies, confirmed that the bound cytochrome c mediated the efficient transfer of electrons from the reducing agent ascorbate to the enzyme. Additionally, pre-steady state analysis at 550nm showed haem a within both covalent complexes catalyzed the oxidation of exogenous ferrocytochroms c. The second order rate constant for these reactions was approximately 25-50 percent of those observed for controls. Our results suggests (i) that electrons are able to enter cytochrome c oxidase via two independent pathways and (ii) that during enzyme turnover the enzyme cycles between two conformers, one of with a substrate binding site at subunit II and the other wth a site at subunits VIa and VIb. Structural analysis suggests that Glu 43, Asp 64 and Glu 83 of subunit VIa and Asp 73, Asp 74 and Glu 78 of subunit VIb are residues that might possibly be involved at the latter site. (AU)