We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.

SummaryThe Tat ( twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.

Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twinarginine motif in the targeting peptide. The translocase component is encoded in Escherichia coli by an unassigned reading frame, yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-arginine translocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.

The enterobacterium Escherichia coli synthesizes two H 2 uptake enzymes, Hyd-1 and Hyd-2. We show using precise electrochemical kinetic measurements that the properties of Hyd-1 and Hyd-2 contrast strikingly, and may be individually optimized to function under distinct environmental conditions. Hyd-2 is well suited for fast and efficient catalysis in more reducing environments, to the extent that in vitro it behaves as a bidirectional hydrogenase. In contrast, Hyd-1 is active for H 2 oxidation under more oxidizing conditions and cannot function in reverse. Importantly, Hyd-1 is O 2 tolerant and can oxidize H 2 in the presence of air, whereas Hyd-2 is ineffective for H 2 oxidation under aerobic conditions. The results have direct relevance for physiological roles of Hyd-1 and Hyd-2, which are expressed in different phases of growth. The properties that we report suggest distinct technological applications of these contrasting enzymes.Hydrogenases catalyze the reversible cleavage of H 2 into protons and electrons, and play an important role in the energy metabolism of a broad range of microorganisms (1). Hydrogenases are classified according to their active site metal ion content, and three phylogenetically distinct classes have so far been identified: di-iron [FeFe]-, nickel-iron [NiFe]-, and mono-iron [Fe]-hydrogenases (1). Nickel-iron hydrogenases are the most abundant of the three types (1), and many members of this class are membrane bound, with the membrane-extrinsic domain consisting of a large subunit containing the active site, and a small subunit accommodating one to three electron-transferring iron-sulfur clusters. The active sites of [NiFe]-hydrogenases contain a nickel atom coordinated by four cysteine-S ligands, two of which bridge to an iron atom that is further coordinated by three unusual diatomic ligands, two cyanides and one carbonyl (2).Hydrogenases are inactivated by O 2

Significance The isolation of an active formate hydrogenlyase is a breakthrough in understanding the molecular basis of bacterial hydrogen production. For over 100 years, Escherichia coli has been known to evolve H 2 when cultured under fermentative conditions. Glucose is metabolized to formate, which is then oxidized to CO 2 with the concomitant reduction of protons to H 2 by a single complex called formate hydrogenlyase, which had been genetically, but never biochemically, characterized. In this study, innovative molecular biology and electrochemical experiments reveal a hydrogenase enzyme with the unique ability to sustain H 2 production even under high partial pressures of H 2 . Harnessing bacterial H 2 production offers the prospect of a source of fully renewable H 2 energy, freed from any dependence on fossil fuel.

The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niμ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases.

The Escherichia coli twin‐arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy‐transducing inner membrane. Complex cofactor‐containing Tat substrates, such as the model (NiFe) hydrogenase‐2 and trimethylamine N‐oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site‐directed mutagenesis, we highlight here this crucial ‘proofreading’ or ‘quality control’ activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor‐insertion activities of the TorA‐specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin‐arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.

An important clue to the mechanism for O(2) tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron-sulfur cluster that is proximal to the active site. The O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron-sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O(2).