ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A Ê ) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O 2 /H 2 O gate with a putative protein channel. This is the ®rst crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme±substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A ؉ B 3 A⅐B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex. PDZ4 domains are found in a few hundred human proteins, either as a single domain or in arrays. These domains mediate binding to other proteins and in this way play important roles in scaffolding and signal transduction (1, 2). Structural studies have shown that the PDZ domains usually bind to the C terminus of their target proteins. A number of crystal and NMR structures of PDZ domains have been solved both with and without bound peptide (for example, Refs 3-6) ( Fig. 1). A wealth of data on different peptides binding to different PDZ domains has been obtained by screening (for example, Refs. 7 and 8) and selection (for example, Refs. 9 and 10). Such studies and those using the yeast twohybrid technique (for example, Ref. 11) provide important information on possible cellular targets for distinct PDZ domains as well as the specificity of the interaction. Moreover, theory and NMR experiments have suggested that the dynamics of PDZ domains and the residues outside of the binding pocket influence their interaction with ligands (12, 13). Despite considerable effort to clarify the structural basis for the PDZ-ligand interaction, only a handful of studies have assessed the binding energetics and specificity of PDZ-peptide interactions using proper equilibrium assays in solution (3, 11, 14 -24). Kinetics of chemical reactions not only provide "end point data" such as equilibrium constants but also yield microscopic rate constants and, more importantly, the possibility of elucidating the mechanisms of binding and probing the binding dynamics as well as the properties of the transition state of the reaction. To assess the binding mechanism, we have performed the first kinetic study of PDZ domains in solution using stopped-flow fluorimetry. The PDZ domains chosen were PDZ3 from human PSD-95, one of the most well studied PDZ domains, and the second PDZ domain from mouse protein tyrosine phosphatase-L1 (PTP-BL; also known...

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To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The K m determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under presteady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.Keywords : cytochrome-c oxidase; electrostatic interaction ; ionic-strength dependence; electron transfer; docking.Cytochrome c oxidase is the terminal enzyme of the respira-earlier findings for other redox partners of cytochrome c (Tiede et al., 1993;Williams et al., 1995), we assume that the interactory chain in mitochondria and many prokaryotes (Gennis and Trumpower, 1994; Capaldi, 1990; de Gier et al., 1994). In Para-tion takes place both via clusters of negatively charged residues on the oxidase and via hydrophobic patches. The initial recognicoccus denitrificans, it consists of four subunits with the three largest subunits homologous to the mitochondrially encoded tion would be driven by the shape and the potential of the negative cluster, and would work long-range, whereas the hydrophoones, while the function of subunit IV is still not evident . Haem a and the binuclear centre (haem bic interaction acting short-range would fine-adjust the redox partners for optimal electron transfer. a 3 · Cu B ) where oxygen reduction takes place reside in subunit I. The Cu A centre acting as the entry point for electrons donated From inspection of the crystal structures of oxidase (Iwata et al., 1995; Tsukihara et al., 1995 Tsukihara et al., , 1996, we were able to make by reduced cytochrome c located in subunit II, and a large body of evidence has shown this subunit to bind cytochrome c. predictions about the putative binding site. We constructed a collection of single mutants (subunit II ; E126Q, D135N, D159N, Studies with modified cytochrome c indicated a number of lysine residues surrounding the haem edge t...

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. K M and k cat values and the bimolecular rate constant were determined under steady-or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the K M remains largely unchanged. Rather, the k cat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.Electron transfer between redox proteins has been under extensive investigation during the last years. The protein surfaces involved in complex formation have been of particular interest as it became clear that electron transfer depends on specific recognition with an affinity low enough to allow rapid dissociation. Several studies with cytochrome c and its different redox partners demonstrated the involvement of electrostatic interactions mediated by lysines surrounding the heme edge on cytochrome c and acidic residues on the counterpart (1-6). Nevertheless, it became obvious from ionic strength dependence measurements that electrostatic interactions are not the only criterion governing optimal electron transfer (2). The bell-shaped dependence of activity on ionic strength lead to the proposal that at low ionic strength the redox partners lack the conformational flexibility to achieve a configuration optimal for electron transfer (7,8) besides the fact that product dissociation becomes rate-limiting. At high ionic strength the rate of non-productive collisions increases due to the shielding of charges. Around the ionic strength optimum, when long range electrostatic forces have roughly aligned the reacting proteins, configurational freedom still prevails for short range forces such as hydrophobic interaction to contribute significantly to a configuration optimal for electron transfer.Cytochrome-c oxidase (cytochrome aa 3 ; EC 1.9.3.1) (for reviews see Refs. 9 -11) is one of the terminal enzymes in the respiratory chain of Paracoccus denitrificans besides a quinol oxidase (cytochrome ba 3 (12)) and an alternative cytochrome oxidase (cytochrome cbb 3 (11)). The three main subunits of cytochrome aa 3 show significant homology to the mitochondrially encoded subunits of the eukaryotic cytochrome oxidase. Heme a and the binuclear center (heme a 3 ⅐Cu B ) are located in subunit I, whereas the Cu A center which is the primary electron acceptor resides in subunit II. As stated above, the involvement of electrostatic interactions in the reaction between cytochrome c and cytochrome-c oxidase mediated by acidic residues predominantly located on subunit II of the P. denitrificans oxidase has already been demonstrated (6).To investigate the c...