Biogenic and Synthetic MnO2 Nanoparticles: Size and Growth Probed with Absorption and Raman Spectroscopies and Dynamic Light Scattering.

MnO2 nanoparticles, similar to those found in soils and sediments, have been characterized via their UV-visible and Raman spectra, combined with dynamic light scattering and reactivity measurements. Synthetic colloids were prepared by thiosulfate reduction of permanganate, their sizes controlled with adsorbates acting as capping agents: bicarbonate, phosphate, and pyrophosphate. Biogenic colloids, products of the manganese oxidase, Mnx, were similarly characterized. The band-gap energies of the colloids were found to increase with decreasing hydrodynamic diameter, Dh, and were proportional to 1/ Dh2, as predicted from quantum confinement theory. The intensity ratio of the two prominent Mn-O stretching Raman bands also varied with particle size, consistent with the ratio of edge to bulk Mn atoms. Reactivity of the synthetic colloids toward reduction by Mn2+, in the presence of pyrophosphate to trap the Mn3+ product, was proportional to the surface to volume ratio, but showed surprising complexity. There was also a remnant unreactive fraction, likely attributable to Mn(III)-induced surface passivation. The band gap was similar for biogenic and synthetic colloids of similar size, but decreased when the enzyme solution contained pyrophosphate, which traps the intermediate Mn(III) and slows MnO2 growth. The band gap/size correlation was used to analyze the growth of the enzymatically produced MnO2 oxides.

Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Förster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase.

Previous studies of a thermophilic alcohol dehydrogenase (ht-ADH) demonstrated a range of discontinuous transitions at 30 °C that include catalysis, kinetic isotope effects, protein hydrogen-deuterium exchange rates, and intrinsic fluorescence properties. Using the Förster resonance energy transfer response from a Trp-NADH donor-acceptor pair in T-jump studies of ht-ADH, we now report microsecond protein motions that can be directly related to active site chemistry. Two distinctive transients are observed: a slow, kinetic process lacking a temperature break, together with a faster transient that is only detectable above 30 °C. The latter establishes a link between enzyme activity and microsecond protein motions near the cofactor binding site, in a region distinct from a previously detected protein network that communicates with the substrate binding site. Though evidence of direct dynamical links between microsecond protein motions and active site bond cleavage events is extremely rare, these studies highlight the potential of T-jump measurements to uncover such properties.

Mn(III) species formed by the multi-copper oxidase MnxG investigated by electron paramagnetic resonance spectroscopy.

The multi-copper oxidase (MCO) MnxG from marine Bacillus bacteria plays an essential role in geochemical cycling of manganese by oxidizing Mn2+(aq) to form manganese oxide minerals at rates that are three to five orders of magnitude faster than abiotic rates. The MCO MnxG protein is isolated as part of a multi-protein complex, denoted as Mnx, which includes one MnxG unit and a hexamer of MnxE3F3 subunit. During the oxidation of Mn2+(aq) catalyzed by the Mnx protein complex, an enzyme-bound Mn(III) species was trapped recently in the presence of pyrophosphate (PP) and analyzed using parallel-mode electron paramagnetic resonance (EPR) spectroscopy. Herein, we provide a full analysis of this enzyme-bound Mn(III) intermediate via temperature dependence studies and spectral simulations. This Mnx-bound Mn(III) species is characterized by a hyperfine-coupling value of A(55Mn) = 4.2 mT (corresponding to 120 MHz) and a negative zero-field splitting (ZFS) value of D = - 2.0 cm-1. These magnetic properties suggest that the Mnx-bound Mn(III) species could be either six-coordinate with a 5B1g ground state or square-pyramidal five-coordinate with a 5B1 ground state. In addition, as a control, Mn(III)PP is also analyzed by parallel-mode EPR spectroscopy. It exhibits distinctly different magnetic properties with a hyperfine-coupling value of A(55Mn) = 4.8 mT (corresponding to 140 MHz) and a negative ZFS value of D = - 2.5 cm-1. The different ZFS values suggest differences in ligand environment of Mnx-bound Mn(III) and aqueous Mn(III)PP species. These studies provide further insights into the mechanism of biological Mn2+(aq) oxidation.

Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Binuclear Activation Mechanism.

The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO2 biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu2+, the electron acceptor. Indeed the type 1 Cu2+ is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO2 formation from MnO2 nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pKa > 8.6 deprotonation, which is assigned to Mn(II)-bound H2O; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(µ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pKa 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(µ-OH)2Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(µ-O)2Mn(IV) or an oligomer, which subsequently nucleates MnO2 nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper .

Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Coordinated Two-Stage Mn(II)/(III) and Mn(III)/(IV) Mechanism.

The bacterial manganese oxidase MnxG of the Mnx protein complex is unique among multicopper oxidases (MCOs) in carrying out a two-electron metal oxidation, converting Mn(II) to MnO2 nanoparticles. The reaction occurs in two stages: Mn(II) → Mn(III) and Mn(III) → MnO2. In a companion study , we show that the electron transfer from Mn(II) to the low-potential type 1 Cu of MnxG requires an activation step, likely forming a hydroxide bridge at a dinuclear Mn(II) site. Here we study the second oxidation step, using pyrophosphate (PP) as a Mn(III) trap. PP chelates Mn(III) produced by the enzyme and subsequently allows it to become a substrate for the second stage of the reaction. EPR spectroscopy confirms the presence of Mn(III) bound to the enzyme. The Mn(III) oxidation step does not involve direct electron transfer to the enzyme from Mn(III), which is shown by kinetic measurements to be excluded from the Mn(II) binding site. Instead, Mn(III) is proposed to disproportionate at an adjacent polynuclear site, thereby allowing indirect oxidation to Mn(IV) and recycling of Mn(II). PP plays a multifaceted role, slowing the reaction by complexing both Mn(II) and Mn(III) in solution, and also inhibiting catalysis, likely through binding at or near the active site. An overall mechanism for Mnx-catalyzed MnO2 production from Mn(II) is presented.

Copper Binding Sites in the Manganese-Oxidizing Mnx Protein Complex Investigated by Electron Paramagnetic Resonance Spectroscopy.

Manganese-oxide minerals (MnOx) are widely distributed over the Earth's surface, and their geochemical cycling is globally important. A multicopper oxidase (MCO) MnxG protein from marine Bacillus bacteria plays an essential role in producing MnOx minerals by oxidizing Mn2+(aq) at rates that are 3 to 5 orders of magnitude faster than abiotic rates. The MnxG protein is isolated as part of a multiprotein complex denoted as "Mnx" that includes accessory protein subunits MnxE and MnxF, with an estimated stoichiometry of MnxE3F3G and corresponding molecular weight of ≈211 kDa. Herein, we report successful expression and isolation of the MCO MnxG protein without the E3F3 hexamer. This isolated MnxG shows activity for Mn2+(aq) oxidation to form manganese oxides. The complement of paramagnetic Cu(II) ions in the Mnx protein complex was examined by electron paramagnetic resonance (EPR) spectroscopy. Two distinct classes of type 2 Cu sites were detected. One class of Cu(II) site (denoted as T2Cu-A), located in the MnxG subunit, is identified by the magnetic parameters g∥ = 2.320 and A∥ = 510 MHz. The other class of Cu(II) sites (denoted as T2Cu-B) is characterized by g∥ = 2.210 and A∥ = 615 MHz and resides in the putative hexameric MnxE3F3 subunit. These different magnetic properties correlate with the differences in the reduction potentials of the respective Cu(II) centers. These studies provide new insights into the molecular mechanism of manganese biomineralization.

Multicopper manganese oxidase accessory proteins bind Cu and heme.

Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

Mn(II,III) oxidation and MnO2 mineralization by an expressed bacterial multicopper oxidase.

Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine Bacillus spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the mnx (Mn oxidation) genes, including mnxG, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the mnxDEFG expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs.

Temperature-Jump Fluorescence Provides Evidence for Fully Reversible Microsecond Dynamics in a Thermophilic Alcohol Dehydrogenase.

Protein dynamics on the microsecond (µs) time scale were investigated by temperature-jump fluorescence spectroscopy as a function of temperature in two variants of a thermophilic alcohol dehydrogenase: W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent µs decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results, which rule out an ionizing histidine as the origin of the fluorescence quenching, are discussed in the context of a Trp49-containing dimer interface that acts as a conduit for thermally activated structural change within the protein interior.

Mn(II) Binding and Subsequent Oxidation by the Multicopper Oxidase MnxG Investigated by Electron Paramagnetic Resonance Spectroscopy.

The dynamics of manganese solid formation (as MnOx) by the multicopper oxidase (MCO)-containing Mnx protein complex were examined by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (CW) EPR spectra of samples of Mnx, prepared in atmosphere and then reacted with Mn(II) for times ranging from 7 to 600 s, indicate rapid oxidation of the substrate manganese (with two-phase pseudo-first-order kinetics modeled using rate coefficients of: k(1obs) = 0.205 ± 0.001 s(-1) and k(2obs) = 0.019 ± 0.001 s(-1)). This process occurs on approximately the same time scale as in vitro solid MnOx formation when there is a large excess of Mn(II). We also found CW and pulse EPR spectroscopic evidence for at least three classes of Mn(II)-containing species in the reaction mixtures: (i) aqueous Mn(II), (ii) a specifically bound mononuclear Mn(II) ion coordinated to the Mnx complex by one nitrogenous ligand, and (iii) a weakly exchange-coupled dimeric Mn(II) species. These findings provide new insights into the molecular mechanism of manganese mineralization.

Ultrafast charge transfer in nickel phthalocyanine probed by femtosecond Raman-induced Kerr effect spectroscopy.

The recently developed technique of femtosecond stimulated Raman spectroscopy, and its variant, femtosecond Raman-induced Kerr effect spectroscopy (FRIKES), offer access to ultrafast excited-state dynamics via structurally specific vibrational spectra. We have used FRIKES to study the photoexcitation dynamics of nickel(II) phthalocyanine with eight butoxy substituents, NiPc(OBu)8. NiPc(OBu)8 is reported to have a relatively long-lived ligand-to-metal charge-transfer (LMCT) state, an essential characteristic for efficient electron transfer in photocatalysis. Following photoexcitation, vibrational transitions in the FRIKES spectra, assignable to phthalocyanine ring modes, evolve on the femtosecond to picosecond time scales. Correlation of ring core size with the frequency of the ν10 (asymmetric C-N stretching) mode confirms the identity of the LMCT state, which has a ∼500 ps lifetime, as well as that of a precursor d-d excited state. An even earlier (∼0.2 ps) transient is observed and tentatively assigned to a higher-lying Jahn-Teller-active LMCT state. This study illustrates the power of FRIKES spectroscopy in elucidating ultrafast molecular dynamics.

Differential control of heme reactivity in alpha and beta subunits of hemoglobin: a combined Raman spectroscopic and computational study.

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or ß hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or ß chains, prior to the quaternary R-T and T-R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for ß than for α chains, and pump-probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 µs for ß but not for α hemes, an interval previously shown to be the first step in the R-T transition. In the α chain νFeHis dropped sharply at 20 µs, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.

CO, NO and O2 as Vibrational Probes of Heme Protein Interactions.

The gaseous XO molecules (X = C, N or O) bind to the heme prosthetic group of heme proteins, and thereby activate or inhibit key biological processes. These events depend on interactions of the surrounding protein with the FeXO adduct, interactions that can be monitored via the frequencies of the Fe-X and X-O bond stretching modes, νFeX and νXO. The frequencies can be determined by vibrational spectroscopy, especially resonance Raman spectroscopy. Backbonding, the donation of Fe dπ electrons to the XO π* orbitals, is a major bonding feature in all the FeXO adducts. Variations in backbonding produce negative νFeX/νXO correlations, which can be used to gauge electrostatic and H-bonding effects in the protein binding pocket. Backbonding correlations have been established for all the FeXO adducts, using porphyrins with electron donating and withdrawing substituents. However the adducts differ in their response to variations in the nature of the axial ligand, and to specific distal interactions. These variations provide differing vantages for evaluating the nature of protein-heme interactions. We review experimental studies that explore these variations, and DFT computational studies that illuminate the underlying physical mechanisms.

Electronic structure and ligand vibrations in FeNO, CoNO, and FeOO porphyrin adducts.

The gaseous ligands, CO, NO, and O2 interact with the Fe ion in heme proteins largely via backbonding of Fe electrons to the π* orbitals of the XO (X = C, N, O) ligands. In these FeXO adducts, the Fe-X stretching frequency varies inversely with the X-O stretching frequency, since increased backbonding strengthens the Fe-X bond while weakening the X-O bond. Inverse frequency correlations have been observed for all three ligands, despite differing electronic and geometric structures, and despite variable composition of the "FeX" vibrational mode, in which Fe-X stretching and Fe-X-O coordinates are mixed for bent FeXO adducts. We report experimental data for 5-coordinate Co(II)(NO) porphyrin adducts (isoelectronic with Fe(II)(OO) adducts), and the results of density functional theory (DFT) modeling for 5-coordinate Fe(II)(NO), Co(II)(NO), and Fe(II)(OO) adducts. Inverse ν(MX)/ν(XO) correlations are obtained computationally, using model porphyrins with graded electron-donating and -withdrawing substituents to modulate the backbonding. Computed slopes agree satisfactorily with experiment, provided nonhybrid functionals are used, which avoid overemphasizing high-spin states. The BP86 functional gives correct ground states, a closed-shell singlet for Co(II)(NO) and an open-shell singlet for the isoelectronic Fe(II)(OO), as corroborated by structural data for Co(II)(NO), and the ν(MX)/ν(XO) slope agreement with experiment for both adducts. However, for Fe(II)(OO) adducts, the computed inverse ν(MX)/ν(XO) correlation applies only to porphyrins with electron-donating and withdrawing substituents of moderate strength. For substituents more donating than -CH3, a direct correlation is obtained, the Fe-O and O-O bonds weakening in concert. This effect is ascribed to the dominance of σ bonding via the in-plane dxz(+dz(2))-π* orbital, when electron-donating substituents raise the d orbital energies sufficiently to render backbonding (dyz-π*) unimportant.

His26 protonation in cytochrome c triggers microsecond ß-sheet formation and heme exposure: implications for apoptosis.

Cytochrome c unfolds locally and reversibly upon heating at pH 3. UV resonance Raman (UVRR) spectra reveal that instead of producing unordered structure, unfolding converts turns and some helical elements to ß-sheet. It also disrupts the Met80-heme bond, and has been previously shown to induce peroxidase activity. Aromatic residues that are H-bonded to a heme propionate (Trp59 and Tyr48) alter their orientation, indicating heme displacement. T-jump/UVRR measurements give time constants of 0.2, 3.9, and 67 µs for successive phases of ß-sheet formation and concomitant reorientation of Trp59. UVRR spectra reveal protonation of histidines, and specifically of His26, whose H-bond to Pro44 anchors the 40s Ω loop; this loop is known to be the least stable 'foldon' in the protein. His26 protonation is proposed to disrupt its H-bond with Pro44, triggering the extension of a short ß-sheet segment at the 'neck' of the 40s Ω loop into the loop itself and back into the 60s and 70s helices. The secondary structure change displaces the heme via H-bonds from residues in the growing ß-sheet, thereby exposing it to exogenous ligands, and inducing peroxidase activity. This unfolding mechanism may play a role in cardiolipin peroxidation by cyt c during apoptosis.

Heme reactivity is uncoupled from quaternary structure in gel-encapsulated hemoglobin: a resonance Raman spectroscopic study.

Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, ν(FeHis), is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving ν(FeHis) frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and reformation of E and F interhelical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.

The molecular biogeochemistry of manganese(II) oxidation.

Micro-organisms capable of oxidizing the redox-active transition metal manganese play an important role in the biogeochemical cycle of manganese. In the present mini-review, we focus specifically on Mn(II)-oxidizing bacteria. The mechanisms by which bacteria oxidize Mn(II) include a two-electron oxidation reaction catalysed by a novel multicopper oxidase that produces Mn(IV) oxides as the primary product. Bacteria also produce organic ligands, such as siderophores, that bind to and stabilize Mn(III). The realization that this stabilized Mn(III) is present in many environments and can affect the redox cycles of other elements such as sulfur has made it clear that manganese and the bacteria that oxidize it profoundly affect the Earth's biogeochemistry.

Multicopper oxidase involvement in both Mn(II) and Mn(III) oxidation during bacterial formation of MnO(2).

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO(2) formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria indicates that multicopper oxidases (MCOs) are required for MnO(2) formation. However, MCOs catalyze one-electron oxidations, whereas the conversion of Mn(II) to MnO(2) is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O(2) and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO(2) also depends on O(2) and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, which is indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO(2) formation.

Probing domain interactions in soluble guanylate cyclase.

Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat ß1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the ß1 H-NOX construct (residues 1-194), the ß1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1ß1 sGC heterodimer (ß1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.

Reversible heme-dependent regulation of human cystathionine ß-synthase by a flavoprotein oxidoreductase.

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS.