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Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key intermediate in H-cluster maturation.

Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes-HydE, HydF, and HydG-in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe(2+), l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)](-) intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)](-) species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe(2+), Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN(-), and the identification of the final organometallic products of the reaction.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.

Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.

The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic (13)C hyperfine interactions for the two CN(-) ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.8 for the Hox form of hydrogenase to

Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.

Cell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school laboratory course. Ninety students in a biotechnology class used CFPS to study the effects of the concentrations of amino acids, cell extract, DNA, and the energy source on accumulation of active super-folder green fluorescent protein. Students estimated product concentrations simply by comparing solution colors to a printed green color gradient. This simple and inexpensive method allows for immediate measurements, and 26 of the 30 groups observed measurable product concentrations within 60 min. These student-generated data were then discussed to illustrate concepts of data analysis such as outliers and standard deviation. We also combined the laboratory experience with a visit to a university campus that included a laboratory tour and a college-style lecture. Our overall objective was to excite the students about the scientific enterprise and to instill a sense of personal relevance and attainability so that these students could realistically consider technical careers.

One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] ([K.sub.m.sup.ATP], 27 [+ or -] 4 [micro]M; [K.sub.m.sup.GTP], 14 [+ or -] 2 [micro]M). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level [K.sub.m.sup.ATP] is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37[degrees]C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

Results demonstrate that the active-site CXXC motifs of the dipeptide DsbA and DsbC are involved in the folding of the soluble Dsb thiol-disulfide oxidoreductases indepedently of the dipeptide's redox potential. The modulation of active sites enhance the disulfide isomerization and influence protein folding and secretion into the periplasmic space.

A study was conducted to show that engineering the disulfide bond machinery of the cell through the expression of periplasmic cysteine oxidoreductases supported the production of active full-length tissue plasminogen activator (tPA). Results indicated several ways for increasing the expression of active, full-length tPA. Findings also correlated the oxidative folding of eukaryotic proteins in the periplasmic space with the isomerization of mismatched disulfides.

An investigation of the in vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC was conducted using genetic selections. Results indicated DsbC acts as a disulfide isomerase. Furthermore, DsbA is a more effective oxidant than DsbC of protein dithiols. Proteins which are effected by DsbC deletions do not have cysteines paired consecutively.

The oxidized state of dsbA periplasmic protein catalyzes disulfide bond formation in proteins. The dsbA protein refolds the misfolded IGF-I by restructuring two disulfide bonds. Disulfide isomerase is the reduction state of dsbA protein. The function of the dsbA protein depends on the redox state of the protein under physiological conditions.