A heme pocket aromatic quadrupole modulates gas binding to cytochrome c'-ß: Implications for NO sensors.

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-ß) resembling that of cytochromes P460. A recent structure of cyt c'-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Bacteroides fragilis expresses three proteins similar to Porphyromonas gingivalis HmuY: Hemophore-like proteins differentially evolved to participate in heme acquisition in oral and gut microbiomes.

Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis to cause dysbiosis also in the gut microbiome.

Computational infrared and Raman spectra by hybrid QM/MM techniques: a study on molecular and catalytic material systems.

Vibrational spectroscopy is one of the most well-established and important techniques for characterizing chemical systems. To aid the interpretation of experimental infrared and Raman spectra, we report on recent theoretical developments in the ChemShell computational chemistry environment for modelling vibrational signatures. The hybrid quantum mechanical and molecular mechanical approach is employed, using density functional theory for the electronic structure calculations and classical forcefields for the environment. Computational vibrational intensities at chemical active sites are reported using electrostatic and fully polarizable embedding environments to achieve more realistic vibrational signatures for materials and molecular systems, including solvated molecules, proteins, zeolites and metal oxide surfaces, providing useful insight into the effect of the chemical environment on the signatures obtained from experiment. This work has been enabled by the efficient task-farming parallelism implemented in ChemShell for high-performance computing platforms.  This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Prevotella intermedia produces two proteins homologous to Porphyromonas gingivalis HmuY but with different heme coordination mode.

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.

Third International Workshop on Improving Data Quality in XAFS Spectroscopy, Q2XAFS2017.

This issue of the Journal of Synchrotron Radiation is a special issue including papers from the Q2XAFS2017 workshop. Here, a brief introduction is given.

Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1.

Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Q(o) site (one of two potential binding sites within cytochrome bc1 using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Q(o) site but bind at the Q(i )site. The discovery that these compounds bind at the Q(i) site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Q(i) also explains the ability of this class to overcome parasite Q(o)-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles.

A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase.

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2-) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2- to the T2Cu centre, but very little is known about how NO2- binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2- may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2- with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.

Distinguishing Nitro vs Nitrito Coordination in Cytochrome c' Using Vibrational Spectroscopy and Density Functional Theory.

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c' (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe-NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.

Hydrogen bonding of the dissociated histidine ligand is not required for formation of a proximal NO adduct in cytochrome c'.

Cytochromes c', that occur in methanotrophic, denitrifying and photosynthetic bacteria, form unusual proximal penta-coordinate NO complexes via a hexa-coordinate distal NO intermediate. Their NO binding properties are similar to those of the eukaryotic NO sensor, soluble guanylate cyclase, for which they provide a valuable structural model. Previous studies suggested that hydrogen bonding between the displaced proximal histidine (His120) ligand (following its dissociation from heme due to trans effects from the distally bound NO) and a conserved aspartate residue (Asp121) could play a key role in allowing proximal NO binding to occur. We have characterized three variants of Alcaligenes xylosoxidans cytochrome c' (AXCP) where Asp121 has been replaced by Ala, Ile and Gln, respectively. In all variants, hydrogen bonding between residue 121 and His120 is abolished yet 5-coordinate proximal NO species are still formed. Our data therefore demonstrate that the His120-Asp121 bond is not essential for proximal NO binding although it likely provides an energy minimum for the displaced His ligand. All variants have altered proximal pocket structure relative to native AXCP.

Lipid-associated aggregate formation of superoxide dismutase-1 is initiated by membrane-targeting loops.

Copper-Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo-SOD1 aggregates may be due to their ability to cause defects within cell membranes by co-aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane-bound copper chaperones. But how apo-SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc-binding loops in apo-SOD1 dimers play a critical role in the binding of 1-octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo-SOD1 monomer also associates with phospholipid bilayer via its zinc-binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane-bound copper chaperone. The orientation also suggests how membrane-bound monomers could act as seeds for membrane-associated SOD1 aggregation.

Fingerprinting redox and ligand states in haemprotein crystal structures using resonance Raman spectroscopy.

It is crucial to assign the correct redox and ligand states to crystal structures of proteins with an active redox centre to gain valid functional information and prevent the misinterpretation of structures. Single-crystal spectroscopies, particularly when applied in situ at macromolecular crystallography beamlines, allow spectroscopic investigations of redox and ligand states and the identification of reaction intermediates in protein crystals during the collection of structural data. Single-crystal resonance Raman spectroscopy was carried out in combination with macromolecular crystallography on Swiss Light Source beamline X10SA using cytochrome c' from Alcaligenes xylosoxidans. This allowed the fingerprinting and validation of different redox and ligand states, identification of vibrational modes and identification of intermediates together with monitoring of radiation-induced changes. This combined approach provides a powerful tool to obtain complementary data and correctly assign the true oxidation and ligand state(s) in redox-protein crystals.

The application of hybrid pixel detectors for in-house SAXS instrumentation with a view to combined chromatographic operation.

This study analyses the potential for laboratory-based size-exclusion chromatography (SEC) integrated small-angle X-ray scattering (SAXS) instrumentation to characterize protein complexes. Using a high-brilliance home source in conjunction with a hybrid pixel X-ray detector, the efficacy of SAXS data collection at pertinent protein concentrations and exposure times has been assessed. Scattering data from SOD1 and from the complex of SOD1 with its copper chaperone, using 10 min exposures, provided data quality in the range 0.03 < q < 0.25 Å(-1) that was sufficient to accurately assign radius of gyration, maximum dimension and molecular mass. These data demonstrate that a home source with integrated SEC-SAXS technology is feasible and would enable structural biologists studying systems containing transient protein complexes, or proteins prone to aggregation, to make advanced preparations in-house for more effective use of limited synchrotron beam time.

Structure of the hypothetical DUF1811-family protein GK0453 from Geobacillus kaustophilus HTA426.

The crystal structure of a conserved hypothetical protein, GK0453, from Geobacillus kaustophilus has been determined to 2.2 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.69, c = 64.18 Å. The structure was determined by the molecular-replacement method and was refined to a final R factor of 22.6% (R(free) = 26.3%). Based on structural homology, the GK0453 protein possesses two independent binding sites and hence it may simultaneously interact with two proteins or with a protein and a nucleic acid.

Challenges of sulfur SAD phasing as a routine method in macromolecular crystallography.

The sulfur SAD phasing method allows the determination of protein structures de novo without reference to derivatives such as Se-methionine. The feasibility for routine automated sulfur SAD phasing using a number of current protein crystallography beamlines at several synchrotrons was examined using crystals of trimeric Achromobacter cycloclastes nitrite reductase (AcNiR), which contains a near average proportion of sulfur-containing residues and two Cu atoms per subunit. Experiments using X-ray wavelengths in the range 1.9-2.4 Å show that we are not yet at the level where sulfur SAD is routinely successful for automated structure solution and model building using existing beamlines and current software tools. On the other hand, experiments using the shortest X-ray wavelengths available on existing beamlines could be routinely exploited to solve and produce unbiased structural models using the similarly weak anomalous scattering signals from the intrinsic metal atoms in proteins. The comparison of long-wavelength phasing (the Bijvoet ratio for nine S atoms and two Cu atoms is ~1.25% at ~2 Å) and copper phasing (the Bijvoet ratio for two Cu atoms is 0.81% at ~0.75 Å) for AcNiR suggests that lower data multiplicity than is currently required for success should in general be possible for sulfur phasing if appropriate improvements to beamlines and data collection strategies can be implemented.

Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

Biological X-ray absorption spectroscopy (BioXAS): a valuable tool for the study of trace elements in the life sciences.

Using X-ray absorption spectroscopy (XAS) the binding modes (type and number of ligands, distances and geometry) and oxidation states of metals and other trace elements in crystalline as well as non-crystalline samples can be revealed. The method may be applied to biological systems as a 'stand-alone' technique, but it is particularly powerful when used alongside other X-ray and spectroscopic techniques and computational approaches. In this review, we highlight how biological XAS is being used in concert with crystallography, spectroscopy and computational chemistry to study metalloproteins in crystals, and report recent applications on relatively rare trace elements utilised by living organisms and metals involved in neurodegenerative diseases.

QM/MM Simulations of Protein Crystal Reactivity Guided by MSOX Crystallography: A Copper Nitrite Reductase Case Study.

The recently developed multiple structures from one crystal (MSOX) serial crystallography method can be used to provide multiple snapshots of the progress of enzymatic reactions taking place within a protein crystal. Such MSOX snapshots can be used as a reference for combined quantum mechanical/molecular mechanical (QM/MM) simulations of enzyme reactivity within the crystal. QM/MM calculations are used to identify details of reference states that cannot be directly observed by X-ray diffraction experiments, such as protonation and oxidation states. These reference states are then used as known fixed endpoints for the modeling of reaction paths. We investigate the mechanism of nitrite reduction in an Achromobacter cycloclastes copper nitrite reductase crystal using MSOX-guided QM/MM calculations, identifying the change in nitrite binding orientation with a change in copper oxidation state, and determining the reaction path to the final NO-bound MSOX structure. The results are compared with QM/MM simulations performed in a solvated environment.

Nature of the copper-nitrosyl intermediates of copper nitrite reductases during catalysis.

The design and synthesis of copper complexes that can reduce nitrite to NO has attracted considerable interest. They have been guided by the structural information on the catalytic Cu centre of the widespread enzymes Cu nitrite reductases but the chemically novel side-on binding of NO observed in all crystallographic studies of these enzymes has been questioned in terms of its functional relevance. We show conversion of NO2 - to NO in the crystal maintained at 170 K and present 'molecular movies' defining events during enzyme turnover including the formation of side-on Cu-NO intermediate. DFT modelling suggests that both true {CuNO}11 and formal {CuNO}10 states may occur as side-on forms in an enzymatic active site with the stability of the {CuNO}10 side-on form governed by the protonation state of the histidine ligands. Formation of a copper-nitrosyl intermediate thus needs to be accommodated in future design templates for functional synthetic Cu-NiR complexes.

A tetranuclear Cu(I) cluster in the metallochaperone protein CopZ.

Copper trafficking proteins and copper-sensitive regulators are often found to be able to bind multiple Cu(I) ions in the form of Cu(I) clusters. We have determined the high-resolution X-ray crystal structure of an Atx1-like copper chaperone protein from Bacillus subtilis containing a novel tetranuclear Cu(I) cluster. The identities and oxidation states of the cluster ions were established unambiguously by refinement of X-ray energy-dependent anomalous scattering factors. The [Cu(4)(S-Cys)(4)(N-His)(2)] cluster geometry provides new structural insights into not only the binding of multiple cuprous ions by metallochaperones but also protein-associated tetranuclear Cu(I) clusters, including those found in eukaryotic copper-responsive transcription factors.

Structural and biophysical properties of metal-free pathogenic SOD1 mutants A4V and G93A.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS. The A4V variant is responsible for the majority of familial ALS cases in North America, causing rapidly progressing paralysis once symptoms begin and the G93A SOD1 variant is overexpressed in often studied murine models of the disease. Here we report the three-dimensional structures of metal-free A4V and of metal-bound and metal-free G93A SOD1. In the metal-free structures, the metal-binding loop elements are observed to be severely disordered, suggesting that these variants may share mechanisms of aggregation proposed previously for other pathogenic SOD1 proteins.