A molecular description of oxygen and peroxide activation in biological systems is difficult, because electrons liberated during X-ray data collection reduce the active centres of redox enzymes catalysing these reactions. Here we describe an effective strategy to obtain crystal structures for high-valency redox intermediates and present a three-dimensional movie of the X-ray-driven catalytic reduction of a bound dioxygen species in horseradish peroxidase (HRP). We also describe separate experiments in which high-resolution structures could be obtained for all five oxidation states of HRP, showing such structures with preserved redox states for the first time.

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Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P 3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-Å resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO 2 H at ϳpH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 Å (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 Å (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.Because of the historical importance of the serine proteases in studies on enzyme catalysis and continuing medicinal interest in their inhibition, the details of their catalytic mechanism remain of interest. For some time there has been a consensus on the overall sequence of steps and key residues involved (4, 5). Catalysis is initiated by the noncovalent binding of the polypeptide substrate to an active site cleft. After attack by a nucleophilic serine onto the scissile amide bond, the acylation phase of the reaction proceeds via the formation of a tetrahedral oxy-anion intermediate that collapses to form an acylenzyme (ester) complex with concomitant release of the Cterminal product fragment. In the deacylation phase of catalysis, attack of a water molecule onto the ester bond results in a second tetrahedral intermediate that collapses, releasing the N-terminal product fragment and regenerating the vacant enzyme.Pioneering work on the structural biology of the serine protease family (4, 6 -10) led to the concept of a conserved active site catalytic triad formed by active site serine, histidine, and aspartate residues. A crucial role as a general base was postulated for the conserved histidine, i.e. it deprotonates both the nucleophilic serine in the acylation phase and the "hydrolytic" water during the deacylation phase. A hydrogen bond between the acti...

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.