Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

Reduced-Dimensionality Al Nanocrystals: Nanowires, Nanobars, and Nanomoustaches.

Aluminum nanocrystals created by catalyst-driven colloidal synthesis support excellent plasmonic properties, due to their high level of elemental purity, monocrystallinity, and controlled size and shape. Reduction in the rate of nanocrystal growth enables the synthesis of highly anisotropic Al nanowires, nanobars, and singly twinned "nanomoustaches". Electron energy loss spectroscopy was used to study the plasmonic properties of these nanocrystals, spanning the broad energy range needed to map their plasmonic modes. The coupling between these nanocrystals and other plasmonic metal nanostructures, specifically Ag nanocubes and Au films of controlled nanoscale thickness, was investigated. Al nanocrystals show excellent long-term stability under atmospheric conditions, providing a practical alternative to coinage metal-based nanowires in assembled nanoscale devices.

Free ferrous ions sustain activity of mammalian stearoyl-CoA desaturase-1.

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid in a reaction catalyzed by a diiron center. The diiron center is well-coordinated by conserved histidine residues and is thought to remain with the enzyme. However, we find here that SCD1 progressively loses its activity during catalysis and becomes fully inactive after about nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe2+ in cells could regulate SCD1 activity and hence lipid metabolism.

A Dual Catalyst Strategy for Controlling Aluminum Nanocrystal Growth.

The synthesis of Al nanocrystals (Al NCs) is a rapidly expanding field, but there are few strategies for size and morphology control. Here we introduce a dual catalyst approach for the synthesis of Al NCs to control both NC size and shape. By using one catalyst that nucleates growth more rapidly than a second catalyst whose ligands affect NC morphology during growth, one can obtain both size and shape control of the resulting Al NCs. The combination of the two catalysts (1) titanium isopropoxide (TIP), for rapid nucleation, and (2) Tebbe's reagent, for specific facet-promoting growth, yields {100}-faceted Al NCs with tunable diameters between 35 and 65 nm. This dual-catalyst strategy could dramatically expand the possible outcomes for Al NC growth, opening the door to new controlled morphologies and a deeper understanding of earth-abundant plasmonic nanocrystal synthesis.

Plasmon-Generated Solvated Electrons for Chemical Transformations.

Methods for generating solvated electrons─free electrons in solution─have focused primarily on alkali metal ionization or high-energy electrons or photons. Here we report the generation of solvated electrons by exciting the plasmon resonance of Al nanocrystals suspended in solution with visible light. Two chemical reactions were performed: a radical-addition reaction with the spin-trap 2-methyl-2-nitrosopropane, and a model cyclization reaction with the radical clock 6-bromohex-1-ene. A quantum efficiency of at least ∼1.1% for plasmon absorbed photon to solvated electron generation can be inferred from the measured radical clock reaction concentration. This study demonstrates a simple way to generate solvated electrons for driving reductive organic chemical reactions in a quantifiable and controlled manner.

Ligand-Dependent Colloidal Stability Controls the Growth of Aluminum Nanocrystals.

The precise size- and shape-controlled synthesis of monodisperse Al nanocrystals remains an open challenge, limiting their utility for numerous applications that would take advantage of their size and shape-dependent optical properties. Here we pursue a molecular-level understanding of the formation of Al nanocrystals by titanium(IV) isopropoxide-catalyzed decomposition of AlH3 in Lewis base solvents. As determined by electron paramagnetic resonance spectroscopy of intermediates, the reaction begins with the formation of Ti3+-AlH3 complexes. Proton nuclear magnetic resonance spectroscopy indicates isopropoxy ligands are removed from Ti by Al, producing aluminum(III) isopropoxide and low-valent Ti3+ catalysts. These Ti3+ species catalyze elimination of H2 from AlH3 inducing the polymerization of AlH3 into colloidally unstable low-valent aluminum hydride clusters. These clusters coalesce and grow while expelling H2 to form colloidally stable Al nanocrystals. The colloidal stability of the Al nanocrystals and their size is determined by the molecular structure and density of coordinating atoms in the reaction, which is controlled by choice of solvent composition.

Bindings of NO, CO, and O2 to multifunctional globin type dehaloperoxidase follow the 'sliding scale rule'.

Dehaloperoxidase-hemoglobin (DHP), a multifunctional globin protein, not only functions as an oxygen carrier as typical globins such as myoglobin and hemoglobin, but also as a peroxidase, a mono- and dioxygenase, peroxygenase, and an oxidase. Kinetics of DHP binding to NO, CO, and O2 were characterized for wild-type DHP A and B and the H55D and H55V DHP A mutants using stopped-flow methods. All three gaseous ligands bind to DHP significantly more weakly than sperm whale myoglobin (SWMb). Both CO and NO bind to DHP in a one-step process to form a stable six-coordinate complex. Multiple-step NO binding is not observed in DHP, which is similar to observations in SWMb, but in contrast with many heme sensor proteins. The weak affinity of DHP for O2 is mainly due to a fast O2 dissociation rate, in accordance with a longer εN-Fe distance between the heme iron and distal histidine in DHP than that in Mb, and an open-distal pocket that permits ligand escape. Binding affinities in DHP show the same 3-4 orders separation between the pairs NO/CO and CO/O2, consistent with the 'sliding scale rule' hypothesis. Strong gaseous ligand discrimination by DHP is very different from that observed in typical peroxidases, which show poor gaseous ligand selectivity, correlating with a neutral proximal imidazole ligand rather than an imidazolate. The present study provides useful insights into the rationale for DHP to function both as mono-oxygenase and oxidase, and is the first example of a globin peroxidase shown to follow the 'sliding scale rule' hypothesis in gaseous ligand discrimination.

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters.

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Six-Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) Has a Single b Heme and Is Capable of Reducing Metal Ion Complexes and Oxygen.

STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe3+ and Cu2+. Ferrous STEAP1 also reacts readily with O2 through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with ∼80 and ∼20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, KD, was ∼30 µM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.

H-NOX from Clostridium botulinum, like H-NOX from Thermoanaerobacter tengcongensis, Binds Oxygen but with a Less Stable Oxyferrous Heme Intermediate.

Heme nitric oxide/oxygen binding protein isolated from the obligate anaerobe Clostridium botulinum (Cb H-NOX) was previously reported to bind NO with a femtomolar K(D) (Nioche, P. et al. Science 2004, 306, 1550-1553). On the other hand, no oxyferrous Cb H-NOX was observed despite full conservation of the key residues that stabilize the oxyferrous complex in the H-NOX from Thermoanaerobacter tengcongensis (Tt H-NOX) (the same study). In this study, we re-measured the kinetics/affinities of Cb H-NOX for CO, NO, and O2. K(D)(CO) for the simple one-step equilibrium binding was 1.6 × 10(-7) M. The K(D)(NO) of Cb H-NOX was 8.0 × 10(-11) M for the first six-coordinate NO complex, and the previous femtomolar K(D)(NO) was actually an apparent K(D) for its multiple-step NO binding. An oxyferrous Cb H-NOX was clearly observed with a K(D)(O2) of 5.3 × 10(-5) M, which is significantly higher than Tt H-NOX's K(D)(O2) = 4.4 × 10(-8) M. The gaseous ligand binding of Cb H-NOX provides another supportive example for the "sliding scale rule" hypothesis (Tsai, A.-L. et al. Antioxid. Redox Signal. 2012, 17, 1246-1263), and the presence of hydrogen bond donor Tyr139 in Cb H-NOX selectively enhanced its affinity for oxygen.

Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin.

A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K(+) or Na(+) and whether the helical movements that result in the canonical E → C conformational change in other microbial rhodopsins are conserved in DeNaR. Visible absorption maxima of DeNaR in its unphotolyzed (dark) state show an 8 nm difference between Na(+) and K(+) in decyl maltopyranoside micelles, indicating an influence of the cations on the retinylidene photoactive site. In addition, electronic paramagnetic resonance (EPR) spectra of the dark states reveal repositioning of helices F and G when K(+) is replaced with Na(+). Furthermore, the conformational changes assessed by EPR spin-spin dipolar coupling show that the light-induced transmembrane helix movements are very similar to those found in bacteriorhodopsin but are altered by the presence of Na(+), resulting in a new feature, the clockwise rotation of helix F. The results establish the first observation of a cation switch controlling the conformations of a microbial rhodopsin and indicate specific interactions of Na(+) with the half-channels of DeNaR to open an appropriate path for ion translocation.

Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation.

We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.

α-Hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of α-subunits of human HbA with hydrogen peroxide.

α-Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds monomeric α-subunits of human hemoglobin A (HbA) and modulates heme iron oxidation and subunit folding states. Although AHSP·αHb complexes autoxidize more rapidly than HbA, the redox mechanisms appear to be similar. Both metHbA and isolated met-ß-subunits undergo further oxidation in the presence of hydrogen peroxide (H(2)O(2)) to form ferryl heme species. Surprisingly, much lower levels of H(2)O(2)-induced ferryl heme are produced by free met-α-subunits as compared with met-ß-subunits, and no ferryl heme is detected in H(2)O(2)-treated AHSP·met-α-complex at pH values from 5.0 to 9.0 at 23 °C. Ferryl heme species were similarly not detected in AHSP·met-α Pro-30 mutants known to exhibit different rates of autoxidation and hemin loss. EPR data suggest that protein-based radicals associated with the ferryl oxidation state exist within HbA α- and ß-subunits. In contrast, treatment of free α-subunits with H(2)O(2) yields much smaller radical signals, and no radicals are detected when H(2)O(2) is added to AHSP·α-complexes. AHSP binding also dramatically reduces the redox potential of α-subunits, from +40 to -78 mV in 1 m glycine buffer, pH 6.0, at 8 °C, demonstrating independently that AHSP has a much higher affinity for Fe(III) versus Fe(II) α-subunits. Hexacoordination in the AHSP·met-α complex markedly decreases the rate of the initial H(2)O(2) reaction with iron and thus provides α-subunits protection against damaging oxidative reactions.

The selectivity of Vibrio cholerae H-NOX for gaseous ligands follows the "sliding scale rule" hypothesis. Ligand interactions with both ferrous and ferric Vc H-NOX.

Vc H-NOX (or VCA0720) is an H-NOX (heme-nitric oxide and oxygen binding) protein from facultative aerobic bacterium Vibrio cholerae. It shares significant sequence homology with soluble guanylyl cyclase (sGC), a NO sensor protein commonly found in animals. Similar to sGC, Vc H-NOX binds strongly to NO and CO with affinities of 0.27 nM and 0.77 µM, respectively, but weakly to O2. When positioned on a "sliding scale" plot [Tsai, A.-l., et al. (2012) Biochemistry 51, 172-186], the line connecting log K(D)(NO) and log K(D)(CO) of Vc H-NOX can almost be superimposed with that of Ns H-NOX. Therefore, the measured affinities and kinetic parameters of gaseous ligands to Vc H-NOX provide more evidence to validate the "sliding scale rule" hypothesis. Like sGC, Vc H-NOX binds NO in multiple steps, forming first a six-coordinate heme-NO complex at a rate of 1.1 × 10(9) M(-1) s(-1), and then converts to a five-coordinate heme-NO complex at a rate that is also dependent on NO concentration. Although the formation of oxyferrous Vc H-NOX cannot be detected at a normal atmospheric oxygen level, ferrous Vc H-NOX is oxidized to the ferric form at a rate of 0.06 s(-1) when mixed with O2. Ferric Vc H-NOX exists as a mixture of high- and low-spin states and is influenced by binding to different ligands. Characterization of both ferric and ferrous Vc H-NOX and their complexes with various ligands lays the foundation for understanding the possible dual roles in gas and redox sensing of Vc H-NOX.

HAMP domain signal relay mechanism in a sensory rhodopsin-transducer complex.

The phototaxis receptor complex composed of sensory rhodopsin II (SRII) and the transducer subunit HtrII mediates photorepellent responses in haloarchaea. Light-activated SRII transmits a signal through two HAMP switch domains (HAMP1 and HAMP2) in HtrII that bridge the photoreceptive membrane domain of the complex and the cytoplasmic output kinase-modulating domain. HAMP domains, widespread signal relay modules in prokaryotic sensors, consist of four-helix bundles composed of two helices, AS1 and AS2, from each of two dimerized transducer subunits. To examine their molecular motion during signal transmission, we incorporated SRII-HtrII dimeric complexes in nanodiscs to allow unrestricted probe access to the cytoplasmic side HAMP domains. Spin-spin dipolar coupling measurements confirmed that in the nanodiscs, SRII photoactivation induces helix movement in the HtrII membrane domain diagnostic of transducer activation. Labeling kinetics of a fluorescein probe in monocysteine-substituted HAMP1 mutants revealed a light-induced shift of AS2 against AS1 by one-half α-helix turn with minimal other changes. An opposite shift of AS2 against AS1 in HAMP2 at the corresponding positions supports the proposal from x-ray crystal structures by Airola et al. (Airola, M. V., Watts, K. J., Bilwes, A. M., and Crane, B. R. (2010) Structure 18, 436-448) that poly-HAMP chains undergo alternating opposite interconversions to relay the signal. Moreover, we found that haloarchaeal cells expressing a HAMP2-deleted SRII-HtrII exhibit attractant phototaxis, opposite from the repellent phototaxis mediated by the wild-type di-HAMP SRII-HtrII complex. The opposite conformational changes and corresponding opposite output signals of HAMP1 and HAMP2 imply a signal transmission mechanism entailing small shifts in helical register between AS1 and AS2 alternately in opposite directions in adjacent HAMPs.

Free ferrous ions sustain activity of mammalian stearoyl-CoA desaturase-1.

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid and the reaction is catalyzed by a diiron center, which is well-coordinated by conserved histidine residues and is thought to remain with enzyme. However, we find that SCD1 progressively loses its activity during catalysis and becomes fully inactive after nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center, and that the addition of free ferrous ions (Fe 2+ ) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe 2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe 2+ in cells could regulate SCD1 activity, and hence lipid metabolism.

Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2.

Six transmembrane epithelial antigen of the prostate (STEAP) 1-4 are membrane-embedded hemoproteins that chelate a heme prosthetic group in a transmembrane domain (TMD). STEAP2-4, but not STEAP1, have an intracellular oxidoreductase domain (OxRD) and can mediate cross-membrane electron transfer from NADPH via FAD and heme. However, it is unknown whether STEAP1 can establish a physiologically relevant electron transfer chain. Here, we show that STEAP1 can be reduced by reduced FAD or soluble cytochrome b5 reductase that serves as a surrogate OxRD, providing the first evidence that STEAP1 can support a cross-membrane electron transfer chain. It is not clear whether FAD, which relays electrons from NADPH in OxRD to heme in TMD, remains constantly bound to the STEAPs. We found that FAD reduced by STEAP2 can be utilized by STEAP1, suggesting that FAD is diffusible rather than staying bound to STEAP2. We determined the structure of human STEAP2 in complex with NADP+ and FAD to an overall resolution of 3.2 Å by cryo-electron microscopy and found that the two cofactors bind STEAP2 similarly as in STEAP4, suggesting that a diffusible FAD is a general feature of the electron transfer mechanism in the STEAPs. We also demonstrated that STEAP2 reduces ferric nitrilotriacetic acid (Fe3+-NTA) significantly slower than STEAP1 and proposed that the slower reduction is due to the poor Fe3+-NTA binding to the highly flexible extracellular region in STEAP2. These results establish a solid foundation for understanding the function and mechanisms of the STEAPs.

Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site.

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.

A "sliding scale rule" for selectivity among NO, CO, and O2 by heme protein sensors.

Selectivity among NO, CO, and O2 is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (K(D)), the ratios for NO:CO:O2 binding stay roughly the same, 1:~10(3):~10(6), when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a "sliding scale rule" for plots of log(K(D)) versus ligand type that allows predictions of K(D) values if one or two are missing. The predicted K(D) for binding of O2to Ns H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O2 affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate, and imidazolate exert a "leveling" effect on ligand binding affinity. The reported picomolar K(D) for binding of NO to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO K(D) ratio of 1:~10(8). This deviation is explained by a complex, multistep process, in which an initial low-affinity hexacoordinate NO complex with a measured K(D) of ≈54 nM, matching that predicted from the sliding scale rule, is formed initially and then is converted to a high-affinity pentacoordinate complex. This multistep six-coordinate to five-coordinate mechanism appears to be common to all NO sensors that exclude O2 binding to capture a lower level of cellular NO and prevent its consumption by dioxygenation.

Opposite displacement of helix F in attractant and repellent signaling by sensory rhodopsin-Htr complexes.

Two forms of the phototaxis receptor sensory rhodopsin I distinguished by differences in its photoactive site have been shown to be directly correlated with attractant and repellent signaling by the dual-signaling protein. In prior studies, differences in the photoactive site defined the two forms, namely the direction of light-induced proton transfer from the chromophore and the pK(a) of an Asp counterion to the protonated chromophore. Here, we show by both in vivo and in vitro measurements that the two forms are distinct protein conformers with structural similarities to two conformers seen in the light-driven proton transport cycle of the related protein bacteriorhodopsin. Measurements of spontaneous cell motility reversal frequencies, an in vivo measure of histidine kinase activity in the phototaxis system, indicate that the two forms are a photointerconvertible pair, with one conformer activating and the other inhibiting the kinase. Protein conformational changes in these photoconversions monitored by site-directed spin labeling show that opposite structural changes in helix F, distant from the photoactive site, correspond to the opposite phototaxis signals. The results provide the first direct evidence that displacements of helix F are directly correlated with signaling and impact our understanding of the sensory rhodopsin I signaling mechanism and the evolution of diverse functionality in this protein family.