Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. Aerobic metabolism produces a great number of activated oxygen species. These species can react with various targets including proteins. In particular, methionine residues can be oxidized into methionine sulfoxide (MetSO).1 Such modifications can alter the biological properties of the targeted proteins (1). For instance, this likely is the case for the ␣-proteinase inhibitor whose oxidation of a methionine residue decreases its affinity relative to its protease target (2) and also for calmodulin whose methionine oxidation leads to a decrease in the efficiency of activation of plasma membrane (3). On the other hand the fact that methionine modifications can be also restricted to only surface-exposed residues was interpreted as a way to protect cells against the action of reactive oxygen species (4). In vivo a ubiquitous enzyme named peptide methionine sulfoxide reductase (MsrA) exists, which reduces both free and protein bound MetSO (5, 6). The fact that the null mutants of both Escherichia coli and yeast showed increased sensitivity against oxidative damage and that overexpression of MsrA gave higher resistance to hydrogen treatment supports an essential role of MsrA in cell viability (7,8). Thus the important biological role attributed to MsrA in vivo justifies a study of the chemical mechanism of the reduction of MetSO by MsrA. The fact that MsrA activity necessitates a thioredoxin recycling system (9 -11) suggested a cysteine residue in the chemical catalysis. Recently, two groups have shown that mutating the invariant cysteine located in the conserved signature Gly-CysPhe-Trp resulted in total loss of enzyme activity (12, 13). Moreover Lowther et al. (13) presented convincing evidence of involvement of intra-thiol-disulfide exchanges in the catalytic mechanism. Based on their data they formulated a reaction mechanism requiring formation of a covalent tetracoordinate intermediate via a nucleophilic attack by the thiolate of the essential cysteine followed by breakdown of the intermediate by means of two thiol-disulfide exchanges, which leads to release of a methionine and a water molecule. In this mechanism, methionine release only occurs if the disulfide exchange is operative. In the present study we show that in fact the nucleophilic attack of the essential cysteine on MetSO leads to formation of a sulfenic acid enzyme intermediate with a concomitant release of methionine. Return of the active site to a reduced state is achieved in vivo via intra-disulfide exchange reactions involving two other cysteines and then by a thioredoxindependent recycling process. EXPERIMENTAL PROCEDURESSite-directed Mutagenesis, Production, and Purification of Wild Type and Mutant E. coli MsrAs-The E. coli strain ...

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the ␣R237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidizedsemiquinone couple in wild-type ETF (E 1 ) is ؉153 ؎ 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E 2 < ؊250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging ␣Arg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable ϳ200-mV destabilization of the flavin anionic semiquinone (E 2 ‫؍‬ ؊31 ؎ 2 mV, and E 1 ‫؍‬ ؊43 ؎ 2 mV). In addition, reduction to the FAD dihydroquinone in ␣R237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the ␣R237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to ␣R237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.

Phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate to form 1.3-diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP-DHs. Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD. In a previous study carried out on Escherichia coli GraP-DH [Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989, the role of His176 as an activator of the reactivity of Cys149 was studied. Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP-DH from E. coli and Bacillus stearothermophilus. The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated. At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys Ϫ /His ϩ ion pair present in the wild type. No pre-steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when D-[1-2 H]glyceraldehyde-3-phosphate was used as the substrate. Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer. These results hypothesized that a phosphorylating GraP-DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient. This is most likely the case for archaeal Methanothermus fervidus GraP-DH which shares less than 15% amino-acid identity with the bacterial or eukaryotic counterparts. No Cys Ϫ /His ϩ ion pair was detectable. Only one thiolate entity was observed with an apparent pKa of 6.2. This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.Keywords : glyceraldehyde-3-phosphate dehydrogenase ; base catalyst; hydride transfer ; archaea; thiol reactivity.Phosphorylating D-glyceraldehyde-3-phosphate dehydroge-DHs. In particular, the refined crystal structure has been deternase (GraP-DH) is a key enzyme involved in the glycolysis and mined for various sources [3Ϫ6]. The structure of the monomer gluconeogenesis pathways in bacteria and eukaryotes. This tetra-unit could be divided into two domains ; the coenzyme domain meric enzyme catalyses reversibly the oxidative phosphorylation spanning amino acids 1Ϫ148 and 311Ϫ330 contains the canoniof D-glyceraldehyde-3-phosphate (GraP) to 1,3-diphosphoglyc-cal Rossmann fold, and the catalytic domain (amino acids 149Ϫ erate (GriP 2 ) in the presence of cofac...

Crystal structures of several members of the nonphosphorylating CoA-independent aldehyde dehydrogenase (ALDH) family have shown that the peculiar binding mode of the cofactor to the Rossmann fold results in a conformational flexibility for the nicotinamide moiety of the cofactor. This has been hypothesized to constitute an essential feature of the catalytic mechanism because the conformation of the cofactor required for the acylation step is not appropriate for the deacylation step. In the present study, the structure of a reaction intermediate of the E268A-glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, obtained by soaking the crystals of the enzyme/NADP complex with the natural substrate, is reported. The substrate is bound covalently in the four monomers and presents the geometric characteristics expected for a thioacylenzyme intermediate. Control experiments assessed that reduction of the coenzyme has occurred within the crystal. The structure reveals that reduction of the cofactor upon acylation leads to an extensive motion of the nicotinamide moiety with a flip of the reduced pyridinium ring away from the active site without significant changes of the protein structure. This event positions the reduced nicotinamide moiety in a pocket that likely constitutes the exit door for NADPH. Arguments are provided that the structure reported here constitutes a reasonable picture of the first thioacylenzyme intermediate characterized thus far in the ALDH family and that the position of the reduced nicotinamide moiety observed in GAPN is the one suitable for the deacylation step within all of the nonphosphorylating CoA-independent ALDH family.