Hannah S. Shafaat

Bacterial diversity in hyperarid Atacama Desert soils

[1] Surface and subsurface soil samples analyzed for this investigation were collected from the hyperarid Yungay region in the Atacama Desert, Chile. This report details the bacterial diversity derived from DNA and PLFA extracted directly from these extremely desiccated soils. Actinobacteria, Proteobacteria, Firmicutes and TM7 division bacteria were detected. Ninety-four percent of the 16S rRNA genes cloned from these soils belong to the Actinobacteria phylum, and the majority of these were most closely related to the genus Frankia. A 24-hour water activity (a w ) time course showed a diurnal cycle that peaked at 0.52 in the early predawn hours, and ranged from 0.01-0.08 during the day. All measured water activity values were below the levels required for microbial growth or enzyme activity. Total organic carbon (TOC) concentrations were above the limit of detection and below the limit of quantification (i.e., 200 μg/g < TOC < 1000 μg/g), and phospholipid fatty acid (PLFA) concentrations ranged from 2 x 105 to 7 x 10 cell equivalents per gram of soil. Soil extracts analyzed for culturable biomass yielded mostly no growth on R2A media; the highest single extract yielded 47 colony forming units (CFU) per gram of soil.

Applications of a Rapid Endospore Viability Assay for Monitoring UV Inactivation and Characterizing Arctic Ice Cores

ABSTRACT We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by l-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% ± 3.8% and 48.9% ± 4.5%, respectively), while only 27.8% ± 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 ± 19 germinable spores/ml and 369 ± 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% ± 9.3%), and the second core contained 131 ± 4 germinable spores/ml and 162 ± 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% ± 8.8%), whereas only 2 CFU/ml were detected by culturing.

Key hydride vibrational modes in [NiFe] hydrogenase model compounds studied by resonance Raman spectroscopy and density functional calculations.

Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. While many enzymatic states of the [NiFe] hydrogenase have been studied extensively, there are multiple catalytically relevant EPR-silent states that remain poorly characterized. Analysis of model compounds using new spectroscopic techniques can provide a framework for the study of these elusive states within the protein. We obtained optical absorption and resonance Raman (RR) spectra of (dppe)Ni(μ-pdt)Fe(CO)(3) and [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)(3)][BF(4)], which are structural and functional model compounds for the EPR-silent Ni-SI and Ni-R states of the [NiFe] hydrogenase active site. The studies presented here use RR spectroscopy to probe vibrational modes of the active site, including metal-hydride stretching vibrations along with bridging ligand-metal and Fe-CO bending vibrations, with isotopic substitution used to identify key metal-hydride modes. The metal-hydride vibrations are essentially uncoupled and represent isolated, localized stretching modes; the iron-hydride vibration occurs at 1530 cm(-1), while the nickel-hydride vibration is observed at 945 cm(-1). The significant discrepancy between the metal-hydride vibrational frequencies reflects the slight asymmetry in the metal-hydride bond lengths. Additionally, time-dependent density functional theory (TD-DFT) calculations were carried out to obtain theoretical RR spectra of these compounds. On the basis of the detailed comparison of theory and experiment, the dominant electronic transitions and significant normal modes probed in the RR experiments were assigned; the primary transitions in the visible wavelengths represent metal-to-metal and metal-to-ligand charge transfer bands. Inherent properties of metal-hydride vibrational modes in resonance Raman spectra and DFT calculations are discussed together with the prospects of observing such vibrational modes in metal-hydride-containing proteins. Such a combined theoretical and experimental approach may be valuable for characterization of analogous redox states in the [NiFe] hydrogenases.

Hydrogen Bonding of Tryptophan Radicals Revealed by EPR at 700 GHz

Redox-active tryptophans are important in biological electron transfer and redox biochemistry. Proteins can tune the electron transfer kinetics and redox potentials of tryptophan via control of the protonation state and the hydrogen-bond strength. We examine the local environment of two neutral tryptophan radicals (Trp108 on the solvent-exposed surface and Trp48 buried in the hydrophobic core) in two azurin variants. Ultrahigh-field EPR spectroscopy at 700 GHz and 25 T allowed complete resolution of all of the principal components of the g tensors of the two radicals and revealed significant differences in the g tensor anisotropies. The spectra together with (2)H ENDOR spectra and supporting DFT calculations show that the g tensor anisotropy is directly diagnostic of the presence or absence as well as the strength of a hydrogen bond to the indole nitrogen. The approach is a powerful one for identifying and characterizing hydrogen bonds that are critical in the regulation of tryptophan-assisted electron transfer and tryptophan-mediated redox chemistry in proteins.

Spectroscopic Comparison of Photogenerated Tryptophan Radicals in Azurin: Effects of Local Environment and Structure

Tryptophan radicals play a significant role in mediating biological electron transfer. We report the photogeneration of a long-lived, neutral tryptophan radical (Az48W*) from the native residue tryptophan-48 in the hydrophobic core of azurin. The optical absorption, electron paramagnetic resonance, and resonance Raman spectra strongly support the formation of a neutral radical, and the data are consistent with direct electron transfer between tryptophan and the copper(II) center. Spectra of the long-lived Az48W* species are compared to those of a previously studied, solvent-exposed radical at position 108 to identify signatures of tryptophan radicals that are sensitive to the local environment. The absorption maxima of Az48W* display an approximately 23 nm hypsochromic shift in the nonpolar environment. The majority of the resonance Raman frequencies are downshifted by approximately 7 cm(-1) relative to the solvent-exposed radical, and large changes in intensity are observed for some modes. The resonance Raman excitation profiles for Az48W* exhibit distinct maxima within the absorption envelope. Electron paramagnetic resonance spectroscopy yields spectra with partially resolved lines caused by hyperfine couplings; the differences between the coupling constants for the buried and solvent-exposed radical are primarily caused by variations in structure. The insights gained by electronic, vibrational, and magnetic resonance spectroscopy enhance our fundamental understanding of the effects of protein environment on radical properties. Hypotheses for the proton transfer pathway within azurin and a deprotonation rate of approximately 5 x 10(6) s(-1) are proposed.

Nickel-Substituted Rubredoxin as a Minimal Enzyme Model for Hydrogenase.

A simple, functional mimic of [NiFe] hydrogenases based on a nickel-substituted rubredoxin (NiRd) protein is reported. NiRd is capable of light-initiated and solution-phase hydrogen production and demonstrates high electrocatalytic activity using protein film voltammetry. The catalytic voltammograms are modeled using analytical expressions developed for hydrogenase enzymes, revealing maximum turnover frequencies of approximately 20-100 s(-1) at 4 °C with an overpotential of 540 mV. These rates are directly comparable to those observed for [NiFe] hydrogenases under similar conditions. Like the native enzymes, the proton reduction activity of NiRd is strongly inhibited by carbon monoxide. This engineered rubredoxin-based enzyme is chemically and thermally robust, easily accessible, and highly tunable. These results have implications for understanding the enzymatic mechanisms of native hydrogenases, and, using NiRd as a scaffold, it will be possible to optimize this catalyst for application in sustainable fuel generation.

Resonance Raman Characterization of a Stable Tryptophan Radical in an Azurin Mutant

Tryptophan radicals play a significant role in mediating biological electron transfer and catalytic processes. Here, we employ visible and UV resonance Raman, EPR, and absorption spectroscopy along with pH/isotope studies and calculations to probe a neutral closed-shell tryptophan and its oxidized radical counterpart in a modified azurin protein. Comparison of the resonance Raman spectra of the radical and closed-shell species combined with vibrational analysis reveals important structural differences between these two tryptophan species. We experimentally observe a significant reduction in bond order of the pyrrole ring of the radical, as evidenced by a 208 cm(-1) downshift of the W3 mode (predominantly C(2)-C(3) stretch). Analysis of the spectra acquired at acidic pH and in deuterated buffer highlights those vibrational modes of the radical that are sensitive to the hydrogen-bonding environment. The most significant change caused by the deuterated buffer is a 45 cm(-1) downshift of an indole nitrogen displacement mode (W17). Our spectra provide evidence that the radical species is a strong hydrogen bond acceptor, particularly in an acidic environment. Furthermore, the pK(a) for this tryptophan radical must be less than 4.0, which falls below previously reported values for l-tryptophan in aqueous solution. The normal mode assignments of the tryptophan radical help characterize its local environment, conformation, hydrogen bonding, and protonation state within a protein.

Azurin as a Protein Scaffold for a Low-coordinate Nonheme Iron Site with a Small-molecule Binding Pocket.

The apoprotein of Pseudomonas aeruginosa azurin binds iron(II) to give a 1:1 complex, which has been characterized by electronic absorption, Mössbauer, and NMR spectroscopies, as well as X-ray crystallography and quantum-chemical computations. Despite potential competition by water and other coordinating residues, iron(II) binds tightly to the low-coordinate site. The iron(II) complex does not react with chemical redox agents to undergo oxidation or reduction. Spectroscopically calibrated quantum-chemical computations show that the complex has high-spin iron(II) in a pseudotetrahedral coordination environment, which features interactions with side chains of two histidines and a cysteine as well as the C═O of Gly45. In the (5)A(1) ground state, the d(z(2)) orbital is doubly occupied. Mutation of Met121 to Ala leaves the metal site in a similar environment but creates a pocket for reversible binding of small anions to the iron(II) center. Specifically, azide forms a high-spin iron(II) complex and cyanide forms a low-spin iron(II) complex.

Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors: R2lox and R2c Proteins

The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a μ-hydroxo/bis-μ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the μ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Significance Metallocofactors enable enzymes to catalyze difficult reactions that would otherwise not be possible, such as the reduction of oxygen. Nature utilizes a number of different metals, and it is crucial that proteins bind the correct metals to execute their function. Nonetheless, the principles that govern metal specificity in proteins remain poorly understood. Here we use an enzyme that forms a heterodinuclear Mn/Fe cofactor with the same protein ligands in both metal-coordinating positions to study how proteins can differentiate between two such similar metals. We show that the protein is intrinsically capable of site-specific metal discrimination. Surprisingly, specificity is achieved in a stepwise process involving not only fundamental affinity differences, but also chemical maturation upon reaction with molecular oxygen. Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving–Williams series (MnII < FeII < NiII < CoII < CuII > ZnII). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving–Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine–valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.