Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.

Summary Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intra-mitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes, and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.

3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacteriumAquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824 -22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd 2؉forms of the enzyme were determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substratefree enzyme or when either A5P alone or PEP alone binds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one face of the tetramer and those located on the other face. In the absence of metal, the asymmetry is lost. Details of the active site that may be important for catalysis are visible at the high resolution achieved in these structures. Most notably, the shape of the PEPbinding pocket forces PEP to assume a distorted geometry at C-2, which might anticipate the conversion from sp 2 to sp 3 hybridization occurring during intermediate formation and which may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2 PEP , respectively. Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxide ion on the nearby C-2 PEP , thus triggering the beginning of the catalytic cycle.3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS 1 ; phospho-2-dehydro-3-deoxyoctonate aldolase, EC 4.1.2.16) catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate ( Fig. 1) (1). This reaction is the first step in the biosynthesis of 3-deoxy-Dmanno-octulosonate, an essential component of the lipopolysaccharide of all Gram-negative bacteria (2). A reaction very similar to KDO8P synthesis is the formation of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) from erythrose 4-phosphate (E4P) and PEP catalyzed by DAH7P synthase (DAH7PS; phospho-2-dehydro-3-deoxyheptonate aldolase, EC 4.1.2.15), which constitutes the first step of the shikimate pathway (3). Earlier studies have established that KDO8PS and DAH7PS share several mechanistic characteristics. For example, in both enzymes, the condensation step of the reaction is stereospecific, involving the addition of the si face of C-3 PEP to the re face of the A5P/E4P carbonyl (4 -7). In particular, KDO8PS and DAH7PS are two of only four known PEP-utilizing e...

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large ␤␣␤ domain (residues 1-180) and the smaller sheet domain (residues 180 -270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.