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Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O2 activation between the aminoquinol and Cu(I).

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family. [ABSTRACT FROM AUTHOR]

The kinetics of heme acquisition by HasA from Pseudomonas aeruginosa (HasAp) is examined. The slow rate of the loop has shown that the induced-fit mechanism of heme uptake in HasAp is not based on a rapid sampling of the H32 loop between open and closed configurations but, rather, that the H32 loop motions are triggered by the formation of the high-spin heme-HasAp intermediate complex.

The dioxygenation of nitric oxide by oxyheme in globin proteins is a major route for NO detoxification in aerobic biological systems, where the reaction has proceeded through an iron(III)-bound peroxynitrite before homolytic cleavage of the O-O bond to form an iron(IV)-oxo and N[O.sub.2] radical. Resonance Raman spectroscopy and rapid-freeze quench methods are used for identifying the millisecond intermediate as an iron(III)-nitrato complex with a symmetric N[O.sub.2] stretch.

The UV-vis, resonance Raman, and EPR spectroscopic techniques are used to study the anaerobically isolated NsrR from Bacillus subtilis containing a [[4Fe-4S].sup.2+] cluster that reacts with NO to form dinitrosyl iron complexes. The results showed that NsrR adopts a dimeric structure in its [[4Fe-4S].sup.2+] holo form as well as in the apo form and could be used to the understand the mechanism of NO sensing in NsrR.

The resonance Raman data and autophosphorylation assays are used for studying wild-type and Y171F DevS in various heme complexes required for sensing environmental conditions leading to nonreplicating persistence in Mycobacterium tuberculosis. The ability of Y171F mutation to disrupt the on-off switch and render all states with distal ligand inactivity is consistent with Tyr171 model, which is required for distal ligand discrimination, but nonessential for autophosphorylation activity.

The RR characterization of full-length heme based sensor DevS from Mycobacterium tuberculosis as well as truncated proteins consisting of the single GAF A domain and both GAF domains is studied. The results indicate that the GAF B domain increases the specificity, thus suggesting that signal transduction in DevS appears to be mediated by specific hydrogen bond network.

Two yeast heme oxygenase proteins, Hmx1 from Saccharomyces cerevisiae and CaHmx1 form Candida albicans were purified and characterized in terms of their structural, spectroscopic and catalytic properties. The construction of soluble Hmx1 and CaHmx1 with C-terminal truncations confirmed that these yeast heme oxygenases, like their mammalian analogues, possess C-terminal lipophilic peptides that anchor them to the membrane.

The pH dependence of the PHM-catalyzed monooxygenation of dansyl-YVG was studied in two different buffer systems in the pH range of 4-10. It was found that the conformational mobility of the bound substrate relative to the copper-superoxo active species is critical to catalysis and showed presence of an accessible vibrational mode coupling Cu-S motion to the H tunneling probability along the C-O...H...C coordinate.