Solution 1H NMR characterization of the distal H-bond network and the effective axial field in the resting-state, high-spin ferric, substrate-bound complex of heme oxygenase from N. meningitidis

The solution (1)H 1D and 2D NMR spectra of the high-spin ferric, resting-state, substrate-bound complex of heme oxygenase, HO, from the pathological bacterium N. meningitidis have been investigated to assess the prospects for definitive assignment of hyperfine shifted and relaxed residue protons and the interpretation of those shifts in terms of the anisotropy and orientation of the paramagnetic susceptibility tensor, chi. Appropriately tailored 1D/2D NMR data, together with analyses of paramagnetic relaxation and a preliminary estimate of the magnetic anisotropy, reveal a chi that is axially anisotropic and oriented along the Fe-His vector. Together with T(-)(2) dependence of the shifts, Deltachi(ax) yields a zero-field splitting constant, D = 9.1 cm(-)(1), which is expected to serve as a very sensitive probe of H-bond interactions between the iron-ligated water and a series of distal ordered water molecules implicated in the mechanism of HO action. The side chains, Gln49 and His53, involved in the stabilization of catalytically relevant water molecules, were found to exhibit orientations rotated by 180 degrees about the beta-gamma bonds in solution relative to those in the crystal. The implication of these reorientations on the details of the distal H-bond network is discussed. The H-bond donor strengths of Gln 49 and His53 were found to respond appropriately to H-bond donor (water) versus H-bond acceptor (cyanide) iron ligands. Very slow NH exchange for the N-terminal portion of the distal helix suggest that an intrinsically "unstable" distal helix may be valid only for the C-terminal portion.     

Resonance Raman spectroscopic study of nitrophorin 1, a nitric oxide-binding heme protein from Rhodnius prolixus, and its nitrosyl and cyano adducts.

The resonance Raman (RR) spectra of nitrophorin 1 (NP1) from the saliva of the blood-sucking insect Rhodnius prolixus, in the absence and presence of nitric oxide (NO) and in the presence of cyanide (CN(-)), have been studied. The NP1 displayed RR spectra characteristic of six-coordinate high-spin (6cHS) ferric heme at room temperature and six-coordinate low-spin heme (6cLS) at low temperature (77 K). NO and CN(-) each bind to Fe(III), both ligands forming 6cLS complexes with NP1. The Fe(III)-NO stretching and bending vibrational frequencies of nitrosyl NP1 were identified at 591 and 578 cm(-1), respectively, on the basis of 15NO isotope shifts. These frequencies are typical of Fe-NO ferric heme proteins, indicating that the NP1 nitrosyl adduct has typical bond strength. Thus, the small NO release rate displayed by NP1 must be due to other protein interactions. Room and cryogenic temperature (77 K) RR spectroscopy and 13C, 15N, and 13C15N isotope substitutions have been used to determine vibrational mode frequencies associated with the Fe(III)-CN(-) bond for the cyano adducts at 454, 443, 397, and 357 cm(-1). The results were analyzed by normal mode calculations to support the assignment of the modes and to assess the NO and CN(-) binding geometries. The observed isotope shifts for the cyano NP1 are smaller than expected and reveal vibrational coupling of Fe(III)-CN(-) modes with heme modes. We also find that the observed frequencies are consistent with the presence of a nearly linear Fe(III)CN(-) linkage (173 degrees ) coexisting with a population with a bent structure (155 degrees ).     

Laser photoexcitation of NAD(P)H induces reduction of P450 BM3 heme domain on the microsecond time scale

We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads to rapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochrome P450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the heme domain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on the microsecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 for substrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observed rate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependence on carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxide bound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 microM. Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide and the ferric BM3 heme domain (Kd increased to 1404 microM) but enhanced the rate of formation of the FeII-CO complex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)H photoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route to probing the P450 catalytic cycle and its transient intermediates.     

The H93G myoglobin cavity mutant as a versatile template for modeling heme proteins: ferrous, ferric, and ferryl mixed-ligand complexes with imidazole in the cavity

One of the difficulties in preparing accurate ambient-temperature model complexes for heme proteins, particularly in the ferric state, has been the generation of mixed-ligand adducts: complexes with different ligands on either side of the heme. The difference in the accessibility of the two sides of the heme in the H93G cavity mutant of myoglobin (Mb) provides a potential general solution to this problem. To demonstrate the versatility of H93G Mb for the preparation of heme protein models, numerous mixed-ligand adducts of ferrous, ferric, and ferryl imidazole-ligated H93G (H93G(Im) Mb) have been prepared. The complexes have been characterized by electronic absorption and magnetic circular dichroism (MCD) spectroscopy in comparison to analogous derivatives of wild type Mb. The starting ferric H93G(Im) Mb state spectroscopically resembles wild-type ferric Mb as expected for a complex containing a single imidazole in the proximal cavity and water bound on the distal side. Addition of a sixth ligand to ferric H93G(Im) Mb, whether charge neutral (imidazole) or anionic (cyanide and azide), results in formation of six-coordinate low-spin complexes with MCD characteristics similar to those of parallel derivatives of wild-type ferric Mb. Reduction of ferric H93G(Im) Mb and subsequent exposure to either CO, NO, or O2 produces ferrous complexes (deoxy, CO, NO, and O2) that consistently exhibit MCD spectra similar to the analogous ferrous species of wild-type ferrous Mb. Most interestingly, reaction of ferric H93G(Im) Mb with H2O2 results in the formation of a stable high-valent oxoferryl complex with MCD characteristics that are essentially identical to those of oxoferryl wild-type Mb. The generation of such a wide array of mixed-ligand heme complexes demonstrates the efficacy of the H93G Mb cavity mutant as a template for the preparation of heme protein model complexes.     

Investigation of the proton-assisted pathway to formation of the catalytically active, ferryl species of P450s by molecular dynamics studies of P450eryF

The recently determined crystal structure of cytochrome P450eryF (6-deoxyerythronolide B hydroxylase; CYP107A1) in its ferric heme substrate-bound form has been used to address one of the most fundamental unresolved aspects of the mechanism of oxidation common to this ubiquitous family of metabolizing heme proteins, the pathway from the twice reduced dioxygen species to the putative catalytically active ferryl oxygen species. Both of these species are too transient to have been characterized experimentally, and the transformation from one to the other has been only partially characterized. The observed requirement of two protons and the formation of water in this transformation suggests a proton-assisted dioxygen bond cleavage as a plausible pathway. However, this pathway is difficult to establish by experiment alone, and the source of the protons in the largely hydrophobic binding pocket of the P450s remains unclear. In this work we have performed molecular dynamics simulations of the twice reduced dioxygen substrate-bound form of this isozyme in order to (i) determine the plausibility of the proposed pathway to compound I formation, a proton-assisted cleavage of the dioxygen bond, and (ii) investigate the possible source of these protons. The analysis of the molecular dynamics trajectories of this species does indeed provide further evidence for this pathway and points to a source of protons. Specifically, two dynamically stable hydrogen bonds to the distal oxygen atom of the dioxygen ligand, one by the substrate and the other by a bound water, are found, consistent with the proposed proton-assisted cleavage of the bond and formation of water. In addition, an extensive dynamically stable hydrogen bond network is formed that connects the distal oxygen to Glu 360, a well-conserved residue in a channel accessible to solvent that could be the ultimate source of protons. The simulations were done for both a protonated and unprotonated Glu and led to a proposed mechanism of proton transfer by it to the distal oxygen atom. In order to validate the procedures used for the simulation of this transient twice-reduced species, we have used these same procedures to perform molecular dynamics simulations of two other forms of P450eryF, the ferric and ferryl substrate-bound species, and compared the results with experiment. The results for the ferric substrate-bound species were assessed by comparisons to the experimentally determined X-ray structure and fluctuations, and good agreement was found. The simulations performed for the ferryl substrate-bound species led to the correct prediction of the observed regio- and stereospecific hydroxylation of its natural substrate, 6-deoxyerythronolide B (6-DEB) at the 6S position. The results of these two additional studies lend credibility to the important mechanistic inferences from the simulations of the transient twice reduced dioxygen species: further evidence for a proton-assisted pathway from it to the catalytically active ferryl species and a possible source of the protons.     

Electronic ground states and vibrational frequency shifts of diatomic ligands in heme adducts

DFT calculations were carried out to study heme complexes with diatomic ligand (CO, NO, or O(2)) and trans-imidazole ligand. The optimized electronic ground states of CO, NO, and O(2) adducts are singlet, doublet, and open-shell singlet, respectively. For O(2) adduct, the open-shell singlet is slightly lower in energy than the close-shell singlet. However, important differences are found in optimized structures and vibrational frequencies. Particularly, the trans-imidazole-induced frequency up-shift of the Fe-O(O) stretching mode can be predicted only with the open-shell singlet as ground state. An analysis of normal modes confirms that the up-shifts in the bent (NO and O(2) ) adducts are mainly due to mixing of Fe-X(O) stretching mode with Fe-X-O bending coordinate. Our study of binding mechanism indicates that a secondary source of the upshifts is the diminished weakening of the Fe-X(O) bonds. The Fe-X(O) bond strengths are modulated by σ competition mechanism, which weakens the Fe-X(O) bond and σ-π cooperation mechanism, which only exists in the bent adducts and enforce the Fe-X(O) bond. -     

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies

Summary We applied the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) approach to evaluate relative stability of the extended (flat) and C-shaped (bent) solution conformational forms of the 5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) molecule in aqueous solution. Calculations indicated that both forms have similar free energies in aqueous solution but detailed energy components are different. The bent solution form has lower intramolecular electrostatic and van der Waals interaction energies. The flat form has more favorable solvation free energy and lower contribution from the bond, angle and torsion angle molecular mechanical internal energies. We exploit these results and combine them with known crystallographic data to provide a model for the progressive binding of the mTHF molecule, a natural cofactor of thymidylate synthase (TS), to the complex forming in the TS-catalyzed reaction. We propose that at the time of initial weak binding in the open enzyme the cofactor molecule remains in a close balance between the flat and bent solution conformations, with neither form clearly favored. Later, thymidylate synthase undergoes conformational change leading to the closure of the active site and the mTHF molecule is withdrawn from the solvent. That effect shifts the thermodynamic equilibrium of the mTHF molecule toward the bent solution form. At the same time, burying the cofactor molecule in the closed active site produces numerous contacts between mTHF and protein that render change in the shape of the mTHF molecule. As a result, the bent solution conformer is converted to more strained L-shaped bent enzyme conformer of the mTHF molecule. The strain in the bent enzyme conformation allows for the tight binding of the cofactor molecule to the productive ternary complex that forms in the closed active site, and facilitates the protonation of the imidazolidine N10 atom, which promotes further reaction. *To whom correspondence should be addressed. Fax: +4822-822-5342; E-mail: a.jarmula@nencki.gov.pl     

13C and 15N NMR studies of iron-bound cyanides of heme proteins and related model complexes: sensitive probe for detecting hydrogen-bonding interactions at the proximal and distal sides

Studies of the 13C and 15N NMR paramagnetic shifts of the iron-bound cyanides in the ferric cyanide forms of various heme proteins containing the proximal histidine and related model complexes are reported. The paramagnetic shifts of the 13C and 15N NMR signals of the iron-bound cyanide are not significantly affected by the substitution of the porphyrin side chains. On the other hand, the paramagnetic shifts of both the 13C and 15N NMR signals decrease with an increase in the donor effect of the proximal ligand, and the 13C NMR signal is more sensitive to a modification of the donor effect of the proximal ligand than the 15N NMR signal. With the tilt of the iron-imidazole bond, the paramagnetic shift of the 13C NMR signal increases, whereas that of the 15N NMR signal decreases. The hydrogen-bonding interaction of the iron-bound cyanide with a solvent decreases the paramagnetic shift of both 13C and 15N NMR signals, and the effect is more pronounced for the 15N NMR signal. Data on the 13C and 15N NMR signals of iron-bound cyanide for various heme proteins are also reported and analyzed in detail. Substantial differences in the 13C and 15N NMR shifts for the heme proteins can be explained on the basis of the results for the model complexes and structures around the heme in the heme proteins. The findings herein show that the paramagnetic shift of the 13C NMR signal of the iron-bound cyanide is a good probe to estimate the donor effect of the proximal imidazole and that the ratio of 15N/13C NMR shifts allows the hydrogen-bonding interaction on the distal side to be estimated.     

The hydrogen bond donor and acceptor ability of thioformic acid

The molecular orbital (MP2) and density functional theory calculations (B3LYP) using 6-311++G(2d,2p) basis set have been carried out on adducts of the cis and trans conformers of two tautomeric forms of thioformic acid (TFA) with water. Thirteen adducts of TFA with water have been optimized. Similar calculations have been carried out on adducts of formic acid with water and seven energy minima have been obtained for them. Our findings indicate that the specific interactions with water play an important role in the conformational stability as thiol form of TFA is the most stable form in gas phase, while it is the thione form which is the most stable in 1:1 adduct. The H-bond acceptor ability of S and O at the thiocarbonyl and carbonyl positions in TFA and FA, respectively, has been compared and observed to be only slightly lower in the former. However, the H-bond donor ability of S–H has been observed to be nearly half to that of O–H. The contributors to stabilization energies of adducts are explored by analyzing geometrical variations, atomic charges, and electron delocalizations.     

Structure, electronic properties and catalytic behaviour of an activity-enhancing CYP102A1 (P450(BM3)) variant

The substrate-free crystal structure of a five-mutation directed evolution variant of CYP102A1 (P450(BM3)) with generic activity-enhancing properties ("KT2") has been determined to 1.9-? resolution. There is a close resemblance to substrate-bound structures of the wild-type enzyme (WT). The disruption of two salt bridges that link the G- and I-helices in WT causes conformational changes that break several hydrogen bonds and reduce the angle of the kink in the I-helix where dioxygen activation is thought to take place. The side-chain of a key active site residue, Phe87, is rotated in one molecule of the asymmetric unit, and the side-chains of Phe158 and Phe261 cascade into the orientations found in fatty-acid-bound forms of the enzyme. The iron is out of the porphyrin plane, towards the proximal cysteine. Unusually, the axial water ligand to the haem iron is not hydrogen-bonded to Ala264. The first electron transfer from the reductase domain to the haem domain of substrate-free KT2 is almost as fast as in palmitate-bound WT even though the reduction potential of the haem domain is only slightly more oxidising than that of substrate-free WT. However, NADPH is turned over slowly in the absence of substrate, so the catalytic cycle is gated by a step subsequent to the first electron transfer-a contrast to WT. Propylbenzene binding slightly raises the first electron transfer rate in WT but not in KT2. It is proposed that the generic rate accelerating properties of KT2 arise from the substrate-free form being in a catalytically ready conformation, such that substrate-induced changes to the structure play a less significant role in promoting the first electron transfer than in WT.     

Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA

Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here, we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants, and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm(-1) in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole-bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm(-1) in the six-coordinate ferrous state is proposed to be the ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm(-1) that is broadened and up-shifted compared to the ν(Fe-His) of Rr-CooA (216 cm(-1)). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm(-1) in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm(-1) disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated, and although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.     

Surface-enhanced Raman scattering as a tool to probe cytochrome P450-catalysed substrate oxidation

Surface-enhanced Raman scattering was used as a spectroscopic tool to investigate the changes brought upon cytochrome P450BSß after fatty acid binding. Differences in the spectra of substrate-free and substrate-bound enzyme were observed indicating the potential for this method to be used in the screening of P450 substrates. In particular, the binding characteristics of myristic acid, an inherent substrate, and hydroxylauric acid, a product of fatty acid oxidation, towards P450BSß in the presence of H₂O₂ were investigated. Specific spectral changes could be assigned to changes in the heme environment only for myristic acid, indicating an occurrence of oxidation process characteristic for the enzymatic substrate.     

Electronic structure of ferric heme nitrosyl complexes with thiolate coordination

The effect of trans thiolate ligation on the coordinated nitric oxide in ferric heme nitrosyl complexes as a function of the thiolate donor strength, induced by variation of NH-S(thiolate) hydrogen bonds, is explored. Density functional theory (DFT) calculations (BP86/TZVP) are used to define the electronic structures of corresponding six-coordinate ferric [Fe(P)(SR)(NO)] complexes. In contrast to N-donor-coordinated ferric heme nitrosyls, an additional Fe-N(O) sigma interaction that is mediated by the dz2/dxz orbital of Fe and a sigma*-type orbital of NO is observed in the corresponding complexes with S-donor ligands. Experimentally, this is reflected by lower nu(N-O) and nu(Fe-N) stretching frequencies and a bent Fe-N-O moiety in the thiolate-bound case.     

Alkylamine-ligated H93G myoglobin cavity mutant: a model system for endogenous lysine and terminal amine ligation in heme proteins such as nitrite reductase and cytochrome f

His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, providing a remarkably versatile template for the preparation of model heme complexes. The investigation of model heme adducts is an important way to probe the relationship between coordination structure and catalytic function in heme enzymes. In this study, we have successfully generated and spectroscopically characterized the H93G Mb cavity mutant ligated with less common alkylamine ligands (models for Lys or the amine group of N-terminal amino acids) in numerous heme iron states. All complexes have been characterized by electronic absorption and magnetic circular dichroism spectroscopy in comparison with data for parallel imidazole-ligated H93G heme iron moieties. This is the first systematic spectral study of models for alkylamine- or terminal amine-ligated heme centers in proteins. High-spin mono- and low-spin bis-amine-ligated ferrous and ferric H93G Mb adducts have been prepared together with mixed-ligand ferric heme complexes with alkylamine trans to nitrite or imidazole as heme coordination models for cytochrome c nitrite reductase or cytochrome f, respectively. Six-coordinate ferrous H93G Mb derivatives with CO, NO, and O(2) trans to the alkylamine have also been successfully formed, the latter for the first time. Finally, a novel high-valent ferryl species has been generated. The data in this study represent the first thorough investigation of the spectroscopic properties of alkylamine-ligated heme iron systems as models for naturally occurring heme proteins ligated by Lys or terminal amines.     

A rare example of three abundant conformers in one retro model of the cisplatin-DNA d(GpG) intrastrand cross link. Unambiguous evidence that guanine O6 to carrier amine ligand hydrogen bonding is not important. possible effect of the Lippard base pair step adjacent to the lesion on carrier ligand hydrogen bonding in DNA adducts

Guanine O6 to carrier ligand hydrogen bonding is a central feature of many hypotheses advanced to explain the anticancer activity of cis-type anticancer drugs, cis-PtA(2)X(2) (A(2) = diamine or two amines). Early structural evidence suggested that cis-Pt(NH(3))(2)(d(GpG)) (the cross-link model for the key cisplatin-DNA adduct) and other cis-PtA(2)(d(GpG)) adducts exist exclusively or mainly as the HH1 conformer with head-to-head (HH) bases. The dynamic motion of the d(GpG) in these adducts is too rapid to permit definitive characterization of both the conformation and the H-bonding. Hence, we use retro models having A(2) ligands designed to slow the motion. Here, we employ Me(2)ppz (N,N'-dimethylpiperazine), which lacks NH groups. Me(2)ppz is unique in having sp(3) N-methyl groups directly in the coordination plane, allowing the coexistence of multiple conformers but hindering dynamic motion in Me(2)ppzPt(d(GpG)) and Me(2)ppzPt(GpG) retro models. Dynamic processes are decreased enough in Me(2)ppzPt(d(GpG)) to permit HPLC separation of three abundant forms. After HPLC separation, the three re-equilibrate, proving that the three forms must be conformers and that Me(2)ppz has little influence on conformer distribution. This marks the first reported characterization of three abundant conformers for one cis-PtA(2)(d(GpG)) adduct. From NMR evidence, the Me(2)ppzPt(d(GpG)) HH1 conformer has uncanted bases. Another conformer, one of two recently discovered conformer types, has head-to-tail (HT) bases with Delta chirality. For this Delta HT1 form, several lines of evidence establish that the dinucleotide moieties have essentially identical structures in d(GpG) (and GpG) adducts of Me(2)ppzPt and other cis-PtA(2) complexes. For example, the shifts of the highly structure-sensitive G H8 NMR signals are almost identical for the Delta HT1 form of all adducts. In previous models, the stabilization of the Delta HT1 form could be attributed to G O6 H-bonding to A(2) NH groups. Such H-bonds are not possible for Me(2)ppz. The unambiguous conclusions are that G O6 H-bonding is weak and that neither canting nor H-bonding is essential in HH forms. These two features are present in almost all other small models but are essentially absent in the cross-link base pair (bp) step in duplexes. We conclude from our work that the forces favoring canting and H-bonding are weak, and we hypothesize that steric effects within the Lippard bp step adjacent to this cross-link bp step easily overcome these forces.     

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid-induced Unfolding

Transitions among various heme coordination/spin states, heme environments and protein conformations of human cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it's kind. Our results indicated that the thermal and acid‐induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe‐S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X‐ray structure of P450 2C8 suggested that the specific structure of the β‐bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid‐induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.     

Resonance Raman and infrared spectroscopic studies of high-output forms of human soluble guanylyl cyclase

The allosteric regulator BAY-41-2272 converts the CO adduct of soluble guanylyl cyclase (CO-sGC) enzyme from a low- to high-output form, with respect to production of cGMP. Resonance Raman (RR) and Fourier Transform Infrared (FTIR) spectroscopic techniques are used to show that the CO-sGC exists as major and minor conformers, both having nu(Fe-CO) and nu(C-O) modes characteristic of 6-coordinate species. It is further shown that addition of BAY-41-2272 to the CO adduct induces the transition of some fraction of the initial CO-heme adducts into two new CO-heme complexes, the fractional conversion being dependent on the temperature. One new complex displays vibrational modes characteristic of pentacoordinated CO-adduct, and its formation is not affected by temperature. The second complex, although slightly different from the original CO-adducts, is hexacoordinated, and its formation is facilitated by temperature. The production of substantial amounts of the 5-coordinate CO adduct upon addition of BAY-41-2272, reveals the fact that several out-of-plane heme deformation modes are simultaneously activated, an observation similar to that realized upon NO activation. While the precise nature of these modes will require elucidation by isotopic labeling experiments, by analogy with earlier studies of other heme proteins, several bands associated with modes attributable to peripheral substituent deformations and methine carbon movements are implicated. The documented formation of two new forms upon addition of Bay-41-2272 (a 5-coordinate and a new 6-coordinate form) is discussed with respect to the implications for enzyme activation.     

An investigation of the deprotonation of hydrazone-based receptors on interaction with anion: develop a colorimetric system distinguishing cyanide from anions

Hydrazone-based receptors, containing fluorene-skeleton substituents, in a THF solution, in the presence of fluoride or cyanide, do not only form H-bond complexes, but mainly undergo deprotonation of the N–H fragments, an event, which is signaled by the color change, the ability of the deprotonation is associated with the fluorene skeleton. The deprotonation process is also reversible by the addition of metal ions, while cyanide from fluoride could be distinguished by the addition of copper (II).     

Molecular properties and potential energy surfaces of the cyanides of the groups 1 and 11 metal atoms

Ab initio calculations on the metal (groups 1 and 11) cyanide complexes show two stable configurations for the ground state geometry, a linear cyanide (MCN) and a triangular (MNC) form with an obtuse M-N-C angle. Lithium complex may exist in a linear isocyanide (MNC) form, but it cannot be differentiated from the triangular configuration because of the flatness of the potential energy surface connecting the two isomers. The metal atom and cyano radical are bonded through a strongly ionic configuration (M+CN-) in both geometrical forms. The MNC triangular form is a very floppy structure having one low frequency for the bending mode, whereas the MCN linear form is more rigid. The CN complexes of the alkali atoms have a triangular geometry as the lowest energy conformer, while the noble metal atoms prefer the linear cyanide one. The relative stability of the two isomers, dipole moments, and effective charges are reported in this paper. The essential aspects of the potential energy surfaces for the ground and the first excited states exhibiting a closely avoided crossing are also explained.     

Conformational and stereoelectronic investigation in 1,2-difluoropropane: The gauche effect

The effect of attaching an additional fluorine atom at C-2 in 1-fluoropropane (FP), giving 1,2-difluoropropane (DFP), on its conformational equilibrium, is theoretically evaluated. This substitution causes critical implications on the conformer stabilities of DFP (TG, GT and GG conformations) and the steric and electrostatic interactions should favor the conformer with fluorine atoms trans. However, the gauche effect plays a major role in describing the energies balance in DFP, shifting the equilibrium towards the conformation in which the two fluorine atoms are gauche. The origin of this effect is discussed through an NBO analysis, which allows the evaluation of both classical and non-classical (hyperconjugation and bent bonds) interactions as the prevailing factors governing the conformational equilibrium of molecules containing the 1,2-difluoroethane fragment.