An Improved Force Field for O2, CO and CN Binding to Metalloporphyrins

Parametrization of a molecular-mechanics program to include terms specific for five- and six-coordinate transition metal complexes is applied to heme complexes. The principal new feature peculiar to five and six coordination is a term that represents the effect of electron-pair repulsion modified by the ligand electronegativity and takes into account the different possible structures of complexes. The model system takes into account the structural differences of the fixing centre in the haemoglobin subunits. The customary proximal histidine is added. The macrocycle heme IX is wholly considered in our model. The calculations show clearly that certain conformations of heme IX–histidine models are much more favourable than others for fixing O₂. From the O₂ binding in haemoglobin and myoglobin and in simple Fe porphyrin models it is concluded that the bent O₂ ligand is best viewed as bound superoxide, O₂ ^?. Rotation of axial ligands are practically free. A small modification of the model in both crystal and protein matrix affects the orientation of the ligands in experimental systems.     

Spin Crossover in Nitrito‐Myoglobin as Revealed by Resonance Raman Spectroscopy

The myoglobin (Mb) heme Fe‐O‐N=O and heme Fe‐O‐N=O/2‐nitrovinyl species have been characterized by resonance Raman spectroscopy. In the heme Fe‐O‐N=O species, the bound nitrite ligand is removed by solvent exchange, thus reforming metmyoglobin (metMb). The high‐spin heme Fe‐O‐N=O unit is converted into a low‐spin heme Fe‐O‐N=O/2‐nitrovinyl species that can be reversibly switched between a low‐ and a high‐spin state without removing the bound nitrite ligand, as observed in the case of the heme Fe‐O‐N=O species. This spin‐state change is likely to be accompanied by a general structural rearrangement in the protein‐binding pocket. This example is the first of a globin protein that can reversibly change its metal spin state through an internal perturbation. These findings provide a basis for understanding the structure–function relationship of the spin cross found in other metalloenzymes and Fe^III–porphyrin complexes.     

Systematic DFT study of gas phase and solvated uranyl and neptunyl complexes [AnO2X4]n (An = U, Np; X = F, Cl, OH, n = -2; X = H2O, n = +2)

The six-valent uranyl and neptunyl complexes [An(VI)O2X4]n (An = U, Np; X = F, Cl, OH, n = -2; X = H2O, n = +2) have been studied within the framework of density functional theory. The relative stabilities of the cis and trans isomers, structural properties, charge distribution, and ligand binding energies have been determined using the modified Perdew-Burke-Ernzerhof functional at the all-electron scalar relativistic level. Uranyl and neptunyl complexes with different ligands have been compared in a systematic fashion, demonstrating close similarity of these actinides in oxidation state VI. In addition, the effect of an aqueous solution has been taken into account with the polarizable continuum model COSMO. Computed averaged ligand binding energies permit one to rationalize the observed different stabilities of the title species in aqueous media.     

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.     

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.     

Crystallographic Evidence for a Sterically Induced Ferryl Tilt in a Non‐Heme Oxoiron(IV) Complex that Makes it a Better Oxidant

Oxoiron(IV) units are often implicated as intermediates in the catalytic cycles of non‐heme iron oxygenases and oxidases. The most reactive synthetic analogues of these intermediates are supported by tetradentate tripodal ligands with N‐methylbenzimidazole or quinoline donors, but their instability precludes structural characterization. Herein we report crystal structures of two [Fe^IV(O)(L)]²⁺ complexes supported by pentadentate ligands incorporating these heterocycles, which show longer average Fe–N distances than the complex with only pyridine donors. These longer distances correlate linearly with log k₂′ values for O‐ and H‐atom transfer rates, suggesting that weakening the ligand field increases the electrophilicity of the Fe=O center. The sterically bulkier quinoline donors are also found to tilt the Fe=O unit away from a linear N‐Fe=O arrangement by 10°.     

STERIC EFFECTS IN REACTIONS OF METAL COMPLEXES. THE LIGAND SUBSTITUTION PROCESSES

Abstract

A model for the calculation of the activation energy for the ligand substitution processes in the metal complexes, occuring in the polar solvents, is suggested. The model takes into account sharp repulsion between the ligands at small distances. The structure of the transition state is analysed depending on the sharp repulsion distance between the ligands, symmetry factor α, and position of the incoming group with respect to the outgoing group (cis or trans). It is shown that for certain values of the parameters cis-substitution may be preferable than trans-substitution.     

Theoretical density functional theory studies on interactions of small biologically active molecules with isolated heme group

We present ab-initio density functional theory studies on the interactions of small biologically active molecules, namely NO, CO, O(2), H(2)O, and NO(2) (-) with the full-size heme group. Our results show that the small molecule-iron bond is the strongest in carbonyl and the weakest in nitrite system. Trans influence induced by NO binding to the five-coordinate heme complex is shown. Nitric oxide in the resulting complex might be described as NO(-). The differences among the small ligands of XO type (CO, NO, O(2)), and their distant chemical behavior from H(2)O and NO(2) (-) ligands in binding to the Fe(II) ion, are shown. Moreover, the role of the heme ring as a reservoir of electrons in the studied complexes is invoked. The analysis of the parameters defining the iron-histidine bond indicates that this bond is longer and weaker in nitrosyl and carbonyl complexes than in the other systems. Our findings support the proposed mechanism of soluble guanylate cyclase (sGC) activation and suggest that the first step of sGC activation by CO may be the same as during the activation by NO. Obtained results are then compared with the data concerning smaller model of the heme, the porphyrin complexes, available in the literature.     

Stability of HIV-1 integrase–ligand complexes: the role of coordinating bonds

It is known that the HIV-1 integrase (IN) strand transfer inhibitors include the chelating fragments forming the coordinating bonds with two Mg²⁺ ions placed in the IN active site. The subject of the article is the role of these coordination bonds on stability of ligand–IN complexes. For this purpose, a set of ligand–IN complexes was investigated theoretically and experimentally. The theoretical model is based on the quantum-chemistry calculations of coordinating bonds geometry and energy. Solvent effects were taking into account using the implicit water model and the two-stage calculation scheme developed previously. For the experimental part of our study a set of the ligands was synthesized, and their IC₅₀ values of IN inhibiting have been measured. It is shown that the main contribution to ligand–IN complexes stability is caused by the substitution of water molecules by the ligand in the first coordination sphere of two Mg²⁺ ions, and the change in the polarization energy of the bulk water. It is shown, that acid–base equilibrium and tautomeric forms of the ligands should be taken into account to improve the prediction ability of the theoretical estimations. All these factors are controlled by the chelating fragments of the ligands. It is demonstrated that our theoretical approach based on the consideration of the coordinating bonds allows to separate active ligands (inhibitors) from inactive ones.     

小分子配位对血红素活性影响的原子-键电负性均衡方法研究

应用原子-键电负性均衡方法, 计算了血红素与小分子的配位络合物的电荷分布和Fukui函数. 血红素与氧、水、一氧化碳和一氧化氮结合时, 铁离子电荷转移到配体原子上. 活性中心铁离子的Fukui函数均大于氧和水配体中的配位氧原子, 而小于一氧化碳和一氧化氮配体中的配位碳和配位氮原子的Fukui函数. 从Fukui函数可以得出, 一氧化碳和一氧化氮很难从它们与活性中心血红素结合的配位络合物中解离出来, 而氧和水易于从它们与血红素结合的配位络合物中解离出来, 进而, 血红素可以再与其它配体结合. 血红素与KCN和NaN3抑制剂作用时, 铁离子的Fukui函数均小于与其配位的碳和氮原子, 表明在过氧化氢酶中血红素的活性作用减弱或被抑制.     

Dioxadiaza- and trioxadiaza-macrocycles containing one dibenzofuran unit selective for cadmium

The binding properties of dioxadiaza- ([17](DBF)N2O2) and trioxadiaza- ([22](DBF)N2O3), macrocyclic ligands containing a rigid dibenzofuran group (DBF), to metal cations and structural studies of their metal complexes have been carried out. The protonation constants of these two ligands and the stability constants of their complexes with Ca2+, Ba2+, and Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+, were determined at 298.2 K in methanol-water (1:1, v/v), and at ionic strength 0.10 mol dm-3 in KNO3. The values of the protonation constants of both ligands are similar, indicating that no cavity size effect is observed. Only mononuclear complexes of these ligands with the divalent metal ions studied were found, and their stability constants are lower than expected, especially for the complexes of the macrocycle with smaller cavity size. However, the Cd2+ complex with [17](DBF)N2O2 exhibits the highest value of stability constant for the whole series of metal ions studied, indicating that this ligand reveals a remarkable selectivity for cadmium(II) in the presence of all the metal ions studied, except copper(II), indicating that this ligand reveals a remarkable selectivity for cadmium(II) in the presence of the mentioned metal ions. The crystal structures of H2[17](DBF)N2O3(2+) (diprotonated form of the ligand) and of its cadmium complex were determined by X-ray diffraction. The Cd2+ ion fits exactly inside the macrocyclic cavity exhibiting coordination number eight by coordination to all the donor atoms of the ligand, and additionally to two oxygen atoms from one nitrate anion and one oxygen atom from a water molecule. The nickel(II) and copper(II) complexes with the two ligands were further studied by UV-vis-NIR and the copper(II) complexes also by EPR spectroscopic techniques in solution indicating square-pyramidal structures and suggesting that only one nitrogen and oxygen donors of the ligands are bound to the metal. However an additional weak interaction of the second nitrogen cannot be ruled out.     

Metal-metal interactions as a function of bridging ligand topology: an electrochemical,spectroelectrochemical, and magnetic study on dinuclear Oxo-Mo(V) complexes with various isomers of dihydroxynaphthalene as bridging ligand

Reaction of [MoV(TpMe,Me)(O)Cl2] with 1,3-, 1,5-, 1,6-, 2,6-, and 2,7-dihydroxynaphthalene affords the dinuclear complexes [[Mo(TpMe,Me)(O)Cl]2(mu-C10H6O2)], abbreviated as 1,3-Mo2, 1,5-Mo2, 1,6-Mo2, 2,6-Mo2, and 2,7-Mo2, according to the substitution pattern of the bridging ligand. Electrochemical, UV-vis/NIR spectroscopic, and variable-temperature magnetic susceptibility studies have been used to probe the effects of the bridging-ligand topology on the metal-metal electronic and magnetic interactions. The complexes can be split into two classes according to the properties of the bridging ligands. Complexes 1,3-Mo2, 1,6-Mo2, and 2,7-Mo2 all have bridging ligands that are topologically equivalent to meta-substituted bridging ligands such as 1,3-dihydroxybenzene, in that (i) there is an odd number of C atoms separating the two oxygen atoms, regardless of the pathway that is taken through the ligand skeleton, and (ii) the doubly oxidized from of the bridging ligand is a diradical. These complexes are classified as being "T-meta" (= topologically equivalent to meta). Complexes 1,5-Mo2 and 2,6-Mo2 have bridging ligands that are topologically equivalent to para-substituted groups such as 1,4-dihydroxybenzene, in that (i) there is an even number of C atoms separating the two oxygen atoms, whichever pathway is taken through the ligand skeleton, and (ii) the doubly oxidized form of the bridging ligand is a diamagnetic quinone. These complexes are classified as "T-para". Electrochemical studies show that the comproportionation constants for the Mo(V)/Mo(IV) mixed-valence states of the T-meta complexes are smaller than those for the T-para complexes. Spectroelectrochemical studies show that the Mo(V)/Mo(IV) mixed-valence states of the T-para complexes show pronounced Mo(IV)-->Mo(V) IVCT transitions, whereas those of the T-meta complexes do not show these transitions. Magnetic susceptibility studies show that the T-meta complexes all display ferromagnetic exchange between the metal centers, whereas the T-para complexes all display antiferromagnetic exchange. Thus, both the electronic and the magnetic properties of these complexes show a clear demarcation into two sets according to the bridging-ligand topology.     

The H93G Myoglobin Cavity Mutant as a Versatile Scaffold for Modeling Heme Iron Coordination Structures in Protein Active Sites and Their Characterization with Magnetic Circular Dichroism Spectroscopy

Preparation of heme model complexes is a challenging subject of long-standing interest for inorganic chemists. His93Gly sperm whale myoglobin (H93G Mb) has the proximal His replaced with the much smaller non-coordinating Gly. This leaves a cavity on the proximal side of the heme into which a wide variety of exogenous ligands can be delivered. The end result is a remarkably versatile scaffold for the preparation of model heme adducts to mimic the heme iron coordination structure of native heme proteins. In this review, we first summarize the quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of the H93G Mb cavity mutant that facilitates the preparation of mixed-ligand derivatives. Then we review our use of magnetic circular dichroism and electronic absorption spectroscopy to characterize nitrogen-, oxygen-, and sulfur-donor-ligated H93G Mb adducts with an emphasis on species not easily prepared by other heme model system approaches and those that serve as spectroscopic models for native heme proteins.     

Assignment of the ferriheme resonances of the low-spin complexes of nitrophorins 1 and 4 by (1)H and (13)C NMR spectroscopy: comparison to structural data obtained from X-ray crystallography

In this work we report the assignment of the majority of the ferriheme resonances of low-spin nitrophorins (NP) 1 and 4 and compare them to those of NP2, published previously. It is found that the structure of the ferriheme complexes of NP1 and NP4, in terms of the orientation of the ligand(s), can be determined with good accuracy by NMR techniques in the low-spin forms and that angle plots proposed previously (Shokhirev, N. V.; Walker, F. A. J. Biol. Inorg. Chem. 1998, 3, 581-594) describe the angle of the effective nodal plane of the axial ligands in solution. The effective nodal plane of low-spin NP1, NP4, and NP2 complexes is in all cases of imidazole and histamine complexes quite similar to the average of the His-59 or -57 and the exogenous ligand angles seen in the X-ray crystal structures. For the cyanide complexes of the nitrophorins, however, the effective nodal plane of the axial ligand does not coincide with the actual histidine-imidazole plane orientation. This appears to be a result of the contribution of an additional source of asymmetry, the orientation of one of the zero-ruffling lines of the heme. Probably this effect exists for the imidazole and histamine complexes as well, but because the effect of asymmetry that occurs from planar exogenous axial ligands is much larger than the effect of heme ruffling the effect of the zero-ruffling line can only be detected for the cyanide complexes, where the only ligand plane is that of the proximal histidine. The three-dimensional structures of the three NP-CN complexes, including that of NP2-CN reported herein, confirm the high degree of ruffling of these complexes. There is an equilibrium between the two heme orientations (A and B) that depends on the heme cavity shape and changes somewhat with exogenous axial ligand. The A:B ratio can be much more accurately measured by NMR spectroscopy than by X-ray crystallography.     

A computational study of the electronic structure, bonding, and spectral properties of tripodal tetraamine Co(III) carbonate complexes

Density functional calculations have been carried out on the experimentally characterized Co(III) [Co(N4)(O2CO)]+ carbonate complexes containing a tripodal tetraamine ligand (N4 = tpa, Metpa, Me2tpa, Me3tpa, pmea, pmap, tepa) and also the model [Co(NH3)4(O2CO)]+ system. Calculations on the model species, performed using both gas-phase and solvent-corrected procedures, have revealed that the inclusion of a condensed-phase environment is necessary to obtain generally satisfactory results for the structural and bonding properties in these systems. Using the solvent-corrected approach, the observed trends in structural parameters for the metal-ligand bonds, 59Co chemical shifts, and changes in visible absorption wavelengths have been satisfactorily reproduced for the [Co(N4)(O2CO)]+ complexes. A time-dependent density functional analysis of the electronic excitations indicates that the overall composition and character of the relevant (d-d) transitions remain similar throughout the series, indicating that the changes in the Co-N interactions, associated with the structural variations occurring as the N-donor ligand identity and size change, appear most likely responsible for the particular spectroscopic features displayed by these species. These observations are further supported by molecular orbital and energy decomposition analyses. The results from the present calculations confirm recent findings that the inclusion of a treatment for solvent effects plays a critical role in the computational modelling of coordination complexes involving mixed (anionic and neutral) ligands.     

CD spectra of polynuclear complexes of diimine ligands: theoretical and experimental evidence for the importance of internuclear exciton coupling

We have recently reported on dinuclear complexes Lambda,Lambda-[Co(2)L(2)Cl(2)]CoCl(4) of two novel chiral ligands (1a and 1b) which contain pyridyl-imine chelate groups (Telfer, S. G.; Sato, T.; Kuroda, R. Chem. Commun. 2003, 1064-1065). The absolute configuration of the cobalt(II) centers was unambiguously assigned by X-ray crystallography. However, the sign of the exciton couplets in their CD spectra was opposite to that expected on the basis of the stereochemistry of the metal centers. We present a rationalization of these anomalous spectra in terms of an "internuclear" exciton coupling model which takes into account the coupling of chromophores located on different metal centers. We have performed a series of semiempirical (ZINDO) calculations which provide quantitative support to this model. These findings show that the absolute configuration of the metal centers in a polynuclear complex may be incorrectly assigned on the basis of CD data if internuclear coupling effects are not taken into consideration. We summarize the CD spectral data of number of other chiral polynuclear complexes from the literature, including dinuclear complexes bridged by the 2,2'-bipyrimidine ligand, complexes of the HAT ligand, and dinuclear triple-stranded helicates. The amplitude of the CD spectra of many of these complexes is not additive with the number of chromophores. These anomalous spectra can be accounted for by taking internuclear coupling effects into consideration.     

Redox potentials of oxoiron(IV) porphyrin π-cation radical complexes: participation of electron transfer process in oxygenation reactions

The oxoiron(IV) porphyrin π-cation radical complex (compound I) has been identified as the key reactive intermediate of several heme enzymes and synthetic heme complexes. The redox properties of this reactive species are not yet well understood. Here, we report the results of a systematic study of the electrochemistry of oxoiron(IV) porphyrin π-cation radical complexes with various porphyrin structures and axial ligands in organic solvents at low temperatures. The cyclic voltammogram of (TMP)Fe(IV)O, (TMP = 5,10,15,20-tetramesitylporphyrinate), exhibits two quasi-reversible redox waves at E(1/2) = 0.88 and 1.18 V vs SCE in dichloromethane at -60 °C. Absorption spectral measurements for electrochemical oxidation at controlled potential clearly indicated that the first redox wave results from the (TMP)Fe(IV)O/[(TMP(+?))Fe(IV)O](+) couple. The redox potential for the (TMP)Fe(IV)O/[(TMP(+?))Fe(IV)O](+) couple undergoes a positive shift upon coordination of an anionic axial ligand but a negative shift upon coordination of a neutral axial ligand (imidazole). The negative shifts of the redox potential for the imidazole complexes are contrary to their high oxygenation activity. On the other hand, the electron-withdrawing effect of the meso-substituent shifts the redox potential in a positive direction. Comparison of the measured redox potentials and reaction rate constants for epoxidation of cyclooctene and demethylation of N,N-dimethylanilines enable us to discuss the details of the electron transfer process from substrates to the oxoiron(IV) porphyrin π-cation radical complex in the oxygenation mechanisms.     

Inclusion of the ligand field contribution in a polarizable molecular mechanics: SIBFA-LF

To account for the distortion of the coordination sphere that takes place in complexes containing open-shell metal cations such as Cu(II), we implemented, in sum of interactions between fragments ab initio computed (SIBFA) molecular mechanics, an additional contribution to take into account the ligand field splitting of the metal d orbitals. This term, based on the angular overlap model, has been parameterized for Cu(II) coordinated to oxygen and nitrogen ligands. The comparison of the results obtained from density functional theory computations on the one hand and SIBFA or SIBFA-LF on the other shows that SIBFA-LF gives geometric arrangements similar to those obtained from quantum mechanical computations. Moreover, the geometric improvement takes place without downgrading the energetic agreement obtained from SIBFA. The systems considered are Cu(II) interacting with six water molecules, four ammonia or four imidazoles, and four water plus two formate anions.     

Nickel(II) complexes with different chromospheres containing macrocyclic ligands: spectroscopic and electrochemical studies

The mixed donor tetradentate (L(1)=N(2)O(2)) and pentadentate (L(2)=N(2)O(2)S) ligands have been prepared by the interaction of 1,3-diaminopropane and thiodiglycolic acid with diamine. These ligands possess two dissimilar coordination sites. Different types of complexes were obtained which have different stoichiometry depending upon the type of ligands. Their structural investigation have been based on elemental analysis, magnetic moment and spectral (ultraviolet, infrared, (1)H NMR, (13)C NMR and mass spectroscopy methods). The Ni(II) complexes show magnetic moments corresponding to two unpaired electrons except [Ni(L(1))](NO(3))(2) which is diamagnetic. Ligand field parameters of these complexes were compared. N(2)O(2)S donor ligand complexes show higher values of ligand field parameters, which are used to detect their geometries. The redox properties and stability of the complexes toward oxidation waves explored by cyclic voltammetry are related to the electron-withdrawing or releasing ability of the substituents of macrocyclic ligands moiety. The Ni(II) complexes displayed Ni(II)/Ni(I) couples irreversible waves associated with Ni(III)/Ni(II) process.     

Changes in the heme ligation during folding of a Geobacter sulfurreducens sensor GSU0935

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.