Although deoxythymidylate cannot be provided directly by ribonucleotide reductase, the gene encoding thymidylate synthase ThyA is absent from the genomes of a large number of nonsymbiotic microbes. We show that ThyX (Thy1) proteins of previously unknown function form a large and distinct class of thymidylate synthases. ThyX has a wide but sporadic phylogenetic distribution, almost exclusively limited to microbial genomes lacking thyA. ThyX and ThyA use different reductive mechanisms, because ThyX activity is dependent on reduced flavin nucleotides. Our findings reveal complexity in the evolution of thymidine in present-day DNA. Because ThyX proteins are found in many pathogenic microbes, they present a previously uncharacterized target for antimicrobial compounds.

Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5 -monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth.

Biological reactions in protein complexes involve structural dynamics spanning many orders of magnitude in time. In standard descriptions of catalysis by enzymes, the transition state between reactant and product is reached by thermal, stochastic motion. In the ultrashort time domain, however, the protein moiety and cofactor motions leading to altered conformations can be coherent rather than stochastic in nature. Such coherent motions may play a key role in controlling the accessibility of the transition state and explain the high efficiency of the reaction. Here we present evidence for coherent population transfer to the product state during an ultrafast reaction catalysed by a key enzyme in aerobic organisms. Using the enzyme cytochrome c oxidase aa3 from the bacterium Paracoccus denitrificans, we have studied haem dynamics during the photo-initiated ultrafast transfer of carbon monoxide from haem a3 to CuB by femtosecond spectroscopy. The ground state of the unliganded a3 species is populated in a stepwise manner in time, indicating that the reaction is mainly governed by coherent vibrations (47cm(-1)). The reaction coordinate involves conformational relaxation of the haem group and we suggest that ligand transfer also contributes.

Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed.

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The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.