X-Ray Crystallographic Analysis of NifB with a Full Complement of Clusters: Structural Insights into the Radical SAM-Dependent Carbide Insertion During Nitrogenase Cofactor Assembly.

NifB is an essential radical SAM enzyme required for the assembly of an 8Fe core of the nitrogenase cofactor. Herein, we report the X-ray crystal structures of Methanobacterium thermoautotrophicum NifB without (apo MtNifB) and with (holo MtNifB) a full complement of three [Fe4 S4 ] clusters. Both apo and holo MtNifB contain a partial TIM barrel core, but unlike apo MtNifB, holo MtNifB is fully assembled and competent in cofactor biosynthesis. The radical SAM (RS)-cluster is coordinated by three Cys, and the adjacent K1- and K2-clusters, representing the precursor to an 8Fe cofactor core, are each coordinated by one His and two Cys. Prediction of substrate channels, combined with in silico docking of SAM in holo MtNifB, suggests the binding of SAM between the RS- and K2-clusters and putative paths for entry of SAM and exit of products of SAM cleavage, thereby providing important mechanistic insights into the radical SAM-dependent carbide insertion concomitant with cofactor core formation.

Strategies Towards Capturing Nitrogenase Substrates and Intermediates via Controlled Alteration of Electron Fluxes.

Nitrogenase utilizes an ATP-dependent reductase to deliver electrons to its catalytic component to enable two important reactions: the reduction of N2 to NH4 + , and the reduction of CO to hydrocarbons. The two nitrogenase-based reactions parallel the industrial Haber-Bosch and Fischer-Tropsch processes, yet they occur under ambient conditions. As such, understanding the enzymatic mechanism of nitrogenase is crucial for the future development of biomimetic strategies for energy-efficient production of valuable chemical commodities. Mechanistic investigations of nitrogenase has long been hampered by the difficulty to trap substrates and intermediates relevant to the nitrogenase reactions. Recently, we have successfully captured CO on the Azotobacter vinelandii V-nitrogenase via two approaches that alter the electron fluxes in a controlled manner: one approach utilizes an artificial electron donor to trap CO on the catalytic component of V-nitrogenase in the resting state; whereas the other employs a mismatched reductase component to reduce the electron flux through the system and consequently accumulate CO on the catalytic component of V-nitrogenase. Here we summarize the major outcome of these recent studies, which not only clarified the catalytic relevance of the one-CO (lo-CO) and multi-CO (hi-CO) bound states of nitrogenase, but also pointed to a potential competition between N2 and CO for binding to the same pair of reactive Fe sites across the sulfur belt of the cofactor. Together, these results highlight the utility of these strategies in poising the cofactor at a well-defined state for substrate- or intermediate-trapping via controlled alteration of electron fluxes, which could prove beneficial for further elucidation of the mechanistic details of nitrogenase-catalyzed reactions.

Structural and Mechanistic Insights into CO2 Activation by Nitrogenase Iron Protein.

The Fe protein of nitrogenase catalyzes the ambient reduction of CO2 when its cluster is present in the all-ferrous, [Fe4 S4 ]0 oxidation state. Here, we report a combined structural and theoretical study that probes the unique reactivity of the all-ferrous Fe protein toward CO2 . Structural comparisons of the Azotobacter vinelandii Fe protein in the [Fe4 S4 ]0 and [Fe4 S4 ]+ states point to a possible asymmetric functionality of a highly conserved Arg pair in CO2 binding and reduction. Density functional theory (DFT) calculations provide further support for the asymmetric coordination of O by the "proximal" Arg and binding of C to a unique Fe atom of the all-ferrous cluster, followed by donation of protons by the proximate guanidinium group of Arg that eventually results in the scission of a C-O bond. These results provide important mechanistic and structural insights into CO2 activation by a surface-exposed, scaffold-held [Fe4 S4 ] cluster.

In vivo structures of an intact type VI secretion system revealed by electron cryotomography.

The type VI secretion system (T6SS) is a versatile molecular weapon used by many bacteria against eukaryotic hosts or prokaryotic competitors. It consists of a cytoplasmic bacteriophage tail-like structure anchored in the bacterial cell envelope via a cytoplasmic baseplate and a periplasmic membrane complex. Rapid contraction of the sheath in the bacteriophage tail-like structure propels an inner tube/spike complex through the target cell envelope to deliver effectors. While structures of purified contracted sheath and purified membrane complex have been solved, because sheaths contract upon cell lysis and purification, no structure is available for the extended sheath. Structural information about the baseplate is also lacking. Here, we use electron cryotomography to directly visualize intact T6SS structures inside Myxococcus xanthus cells. Using sub-tomogram averaging, we resolve the structure of the extended sheath and membrane-associated components including the baseplate. Moreover, we identify novel extracellular bacteriophage tail fiber-like antennae. These results provide new structural insights into how the extended sheath prevents premature disassembly and how this sophisticated machine may recognize targets.

Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.

Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid.

Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB.

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical.

Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster.

Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities.IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation.

Probing the coordination and function of Fe4S4 modules in nitrogenase assembly protein NifB.

NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe4S4] modules (designated K1 and K2) into an [Fe8S9C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe4S4] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe4S4] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5'-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron-sulfur (FeS) enzymes.

In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System.

The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche.

Cluster assembly in nitrogenase.

The versatile enzyme system nitrogenase accomplishes the challenging reduction of N2and other substrates through the use of two main metalloclusters. For molybdenum nitrogenase, the catalytic component NifDK contains the [Fe8S7]-core P-cluster and a [MoFe7S9C-homocitrate] cofactor called the M-cluster. These chemically unprecedented metalloclusters play a critical role in the reduction of N2, and both originate from [Fe4S4] clusters produced by the actions of NifS and NifU. Maturation of P-cluster begins with a pair of these [Fe4S4] clusters on NifDK called the P*-cluster. An accessory protein NifZ aids in P-cluster fusion, and reductive coupling is facilitated by NifH in a stepwise manner to form P-cluster on each half of NifDK. For M-cluster biosynthesis, two [Fe4S4] clusters on NifB are coupled with a carbon atom in a radical-SAM dependent process, and concomitant addition of a 'ninth' sulfur atom generates the [Fe8S9C]-core L-cluster. On the scaffold protein NifEN, L-cluster is matured to M-cluster by the addition of Mo and homocitrate provided by NifH. Finally, matured M-cluster in NifEN is directly transferred to NifDK, where a conformational change locks the cofactor in place. Mechanistic insights into these fascinating biosynthetic processes are detailed in this chapter.

Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography.

Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal body. Based on pilin features, T4P are classified into type IVa pili (T4aP) and type IVb pili (T4bP)1,2. T4aP are more widespread and are involved in cell motility3, DNA transfer4, host predation5 and electron transfer6. T4bP are less prevalent and are mainly found in enteropathogenic bacteria, where they play key roles in host colonization7. Following similar work on T4aP machines8,9, here we use electron cryotomography10 to reveal the three-dimensional in situ structure of a T4bP machine in its piliated and non-piliated states. The specific machine we analyse is the Vibrio cholerae toxin-coregulated pilus machine (TCPM). Although only about half of the components of the TCPM show sequence homology to components of the previously analysed Myxococcus xanthus T4aP machine (T4aPM), we find that their structures are nevertheless remarkably similar. Based on homologies with components of the M. xanthus T4aPM and additional reconstructions of TCPM mutants in which the non-homologous proteins are individually deleted, we propose locations for all eight TCPM components within the complex. Non-homologous proteins in the T4aPM and TCPM are found to form similar structures, suggesting new hypotheses for their functions and evolutionary histories.

Architecture of the type IVa pilus machine.

Type IVa pili are filamentous cell surface structures observed in many bacteria. They pull cells forward by extending, adhering to surfaces, and then retracting. We used cryo-electron tomography of intact Myxococcus xanthus cells to visualize type IVa pili and the protein machine that assembles and retracts them (the type IVa pilus machine, or T4PM) in situ, in both the piliated and nonpiliated states, at a resolution of 3 to 4 nanometers. We found that T4PM comprises an outer membrane pore, four interconnected ring structures in the periplasm and cytoplasm, a cytoplasmic disc and dome, and a periplasmic stem. By systematically imaging mutants lacking defined T4PM proteins or with individual proteins fused to tags, we mapped the locations of all 10 T4PM core components and the minor pilins, thereby providing insights into pilus assembly, structure, and function.