Baeyer-Villiger Monooxygenase-Mediated Synthesis of Esomeprazole As an Alternative for Kagan Sulfoxidation.

A wild-type Baeyer-Villiger monooxygenase was engineered to overcome numerous liabilities in order to mediate a commercial oxidation of pyrmetazole to esomeprazole, using air as the terminal oxidant in an almost exclusively aqueous reaction matrix. The developed enzyme and process compares favorably to the incumbent Kagan inspired chemocatalytic oxidation, as esomeprazole was isolated in 87% yield, in >99% purity, with an enantiomeric excess of >99%.

Catalytic effectiveness, a measure of enzyme proficiency for industrial applications.

Recent attention has been paid to the inadequacy of using the ratio Vmax/KM as a measure of enzyme performance, particularly in the context of industrial biocatalysis. This can lead to misleading expectations of enzyme performance and can be troublesome when used to select among different variants for scale-up evaluation under process conditions. To address these issues, we derive the average velocity based on the time-integrated behavior of the enzyme over the course of the reaction. The resulting expression, deemed catalytic effectiveness, captures important features of the system that have heretofore been ignored (such as highly variable substrate and/or product concentrations and inhibition) and offers a rigorous way to compare enzymes for their capacity to carry out industrial transformations.

Peroxo-type intermediates in class I ribonucleotide reductase and related binuclear non-heme iron enzymes.

We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class Ia ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O stretch frequencies, Mossbauer isomer shifts, absorption spectra, J-coupling constants, electron affinities, and free energies of O(2) and proton or water binding are presented for a series of possible intermediates. The results enable structure-property correlations and a new rationale for the changes in carboxylate conformations occurring during the O(2) reaction of this class of non-heme iron enzymes. Our procedure identifies and characterizes various possible candidates for peroxo intermediates experimentally observed along the ribonucleotide reductase dioxygen activation reaction. The study explores how water or a proton can bind to the di-iron site of ribonucleotide reductase and facilitate changes that affect the electronic structure of the iron sites and activate the site for further reaction. Two potential reaction pathways are presented: one where water adds to Fe1 of the cis-mu-1,2 peroxo intermediate P causing opening of a bridging carboxylate to form intermediate P' that has an increased electron affinity and is activated for proton-coupled electron transfer to form the Fe(III)Fe(IV) intermediate X; and one that is more energetically favorable where the P to P' conversion involves addition of a proton to a terminal carboxylate ligand in the site which increases the electron affinity and triggers electron transfer to form X. Both pathways provide a mechanism for the activation of peroxy intermediates in binuclear non-heme iron enzymes for reactivity. The studies further show that water coordination can induce the conformational changes observed in crystal structures of the met state.

Spectroscopy and electronic structures of mono- and binuclear high-valent non-heme iron-oxo systems.

High-valent iron-oxo intermediates are known or believed to be key oxidizing species in the catalytic mechanisms of many mononuclear and binuclear non-heme iron enzymes. So far only limited experimental data on their electronic structures are available. In this study we extend knowledge from the experimentally well characterized mononuclear Fe(IV)=O (S=1) biomimetic model system to computational insight into the spectroscopy and electronic structures of mono-and binuclear high-valent iron-oxo enzyme intermediates. In the mononuclear Fe(IV)=O complexes, we predict the spectroscopy and energies of the electronic transitions to be very different for the S=1 and S=2 spin states, but the iron-oxo bonding for both spin states to be very similar. A comparison of the S=2 mono- and binuclear high-valent iron-sites predicts similar electronic transitions. However, the bent iron-oxo bridge and interactions with the second iron-center in the dimer shift the transitions to higher energies and splits the d(xz/yz) orbital set. These electronic structure and TD-DFT results provide a basis for understanding the spectroscopy and electronic structures of high-valent intermediates in mono- and binuclear non-heme iron enzymes.

Heterodisulfide reductase from Methanothermobacter marburgensis contains an active-site [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction.

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron-sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). In this work, the ground- and excited-state electronic properties of the paramagnetic Fe-S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable-temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe-4S](+) clusters in dithionite-reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe-4S](3+) clusters in oxidized HDR samples treated separately with the CoM-SH and CoB-SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe-4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.

Spectroscopic and electronic structure studies of intermediate X in ribonucleotide reductase R2 and two variants: a description of the FeIV-oxo bond in the FeIII-O-FeIV dimer.

Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the FeIII-FeIV active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the FeIII/FeIV active site, which displayed three FeIV d-d transitions between 16,700 and 22,600 cm(-1), two FeIV d-d spin-flip transitions between 23,500 and 24,300 cm(-1), and five oxo to FeIV and FeIII charge transfer (CT) transitions between 25,000 and 32,000 cm(-1). The FeIV d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-pi manifold. Furthermore, the FeIV d-pi splittings in the WT are too large to correlate with a bis-mu-oxo structure. The assignment of the FeIV d-d transitions in WT intermediate X best correlates with a bridged mu-oxo/mu-hydroxo [FeIII(mu-O)(mu-OH)FeIV] structure. The mu-oxo/mu-hydroxo core structure provides an important sigma/pi superexchange pathway, which is not present in the bis-mu-oxo structure, to promote facile electron transfer from Y122 to the remote FeIV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis.

Comparing the electronic properties of the low-spin cyano-ferric [Fe(N4)(Cys)] active sites of superoxide reductase and p450cam using ENDOR spectroscopy and DFT calculations.

Superoxide reductase (SOR) and P450 enzymes contain similar [Fe(N)4(SCys)] active sites and, although they catalyze very different reactions, are proposed to involve analogous low-spin (hydro)peroxo-Fe(III) intermediates in their respective mechanisms that can be modeled by cyanide binding. The equatorial FeN4 ligation by four histidine ligands in CN-SOR and the heme in CN-P450cam is directly compared by 14N ENDOR, while the axial Fe-CN and Fe-S bonding is probed by 13C ENDOR of the cyanide ligand and 1Hbeta ENDOR measurements to determine the spin density delocalization onto the cysteine sulfur. There are small, but notable, differences in the bonding between Fe(III) and its ligands in the two enzymes. The ENDOR measurements are complemented by DFT computations that support the semiempirical equation used to compute spin densities on metal-coordinated cysteinyl and shed light on bonding changes as the Fe-C-N linkage bends. They further indicate that H bonds to the cysteinyl thiolate sulfur ligand reduce the spin density on the sulfur in both active sites to a degree that exceeds the difference induced by the alternative sets of "in-plane" nitrogen ligands.

Geometries and electronic structures of cyanide adducts of the non-heme iron active site of superoxide reductases: vibrational and ENDOR studies.

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.

Superoxide reductase: fact or fiction?

It was recently proposed that anaerobic microorganisms contain a new pathway for detoxification of reactive oxygen species. This is centered around a novel mononuclear iron-containing enzyme, superoxide reductase (SOR), which catalyzes the reduction, rather than the dismutation, of superoxide to hydrogen peroxide. A surprisingly large amount of relevant data has accumulated in the two years or so since the proposal was made. Herein we address the questions: to what extent has the pathway been validated, and what fundamental issues have yet to be answered in considering the response of anaerobes to reactive oxygen species? The evidence for superoxide reduction by SOR is now overwhelming and comes from a variety of anaerobic and microaerophilic species. Moreover, the available spectroscopic and structural information provide a convincing case that the catalytic Fe site of SOR is structurally and electronically tuned to mediate superoxide reduction rather than oxidation. Kinetic analyses also support the original proposal of NAD(P)H, via rubredoxin and NAD(P)H:rubredoxin oxidoreductase, as the source of reductant. What is still to be determined is the fate of the peroxide generated by the SOR reaction. In particular, the role of otherwise well-characterized proteins like rubrerythrin, NADH peroxidase, and rubredoxin:oxygen oxidoreductase in "anaerobic" oxygen metabolism has yet to be established.

Spectroscopic characterization of the [Fe(His)(4)(Cys)] site in 2Fe-superoxide reductase from Desulfovibrio vulgaris.

The electronic and vibrational properties of the [Fe(His)(4)(Cys)] site (Center II) responsible for catalysis of superoxide reduction in the two-iron superoxide reductase (2Fe-SOR) from Desulfovibrio vulgaris have been investigated using the combination of EPR, resonance Raman, UV/visible/near-IR absorption, CD, and VTMCD spectroscopies. Deconvolution of the spectral contributions of Center II from those of the [Fe(Cys)(4)] site (Center I) has been achieved by parallel investigations of the C13S variant, which does not contain Center I. The resonance Raman spectrum of ferric Center II has been assigned based on isotope shifts for (34)S and (15)N globally labeled proteins. As for the [Fe(His)(4)(Cys)] active site in 1Fe-SOR from Pyrococcus furiosus, the spectroscopic properties of ferric and ferrous Center II in D. vulgaris 2Fe-SOR are indicative of distorted octahedral and square-pyramidal coordination geometries, respectively. Differences in the properties of the ferric [Fe(His)(4)(Cys)] sites in 1Fe- and 2Fe-SORs are apparent in the rhombicity of the S=5/2 ground state ( E/ D=0.06 and 0.28 in 1Fe- and 2Fe-SORs, respectively), the energy of the CysS(-)(p(pi))-->Fe(3+)(d(pi)) CT transition (15150+/-150 cm(-1) and 15600+/-150 cm(-1) in 1Fe- and 2Fe-SORs, respectively) and in changes in the Fe-S stretching region of the resonance Raman spectrum indicative of a weaker Fe-S(Cys) bond in 2Fe-SORs. These differences are interpreted in terms of small structural perturbations in the Fe coordination sphere with changes in the Fe-S(Cys) bond strength resulting from differences in the peptide N-H.S(Cys) hydrogen bonding within a tetrapeptide bidentate "chelate". Observation of the characteristic intervalence charge transfer transition of a cyano-bridged [Fe(III)-NC-Fe(II)(CN)(5)] unit in the near-IR VTMCD spectra of ferricyanide-oxidized samples of both P. furiosus 1Fe-SOR and D. vulgaris 2Fe-SOR has confirmed the existence of novel ferrocyanide adducts of the ferric [Fe(His)(4)(Cys)] sites in both 1Fe- and 2Fe-SORs.

Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase.

Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of Pyrococcus furiosus superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial S = 32 ground state with ED = 0.06 and D = 12 +/- 2 cm(-1) (where D and E are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane NO(-)(pi*)-to-Fe(3+)(dpi) charge-transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl S, as evidenced by the identification of nu(N-O) at 1,721 cm(-1), nu(Fe-NO) at 475 cm(-1), and nu(Fe-S(Cys), at 291 cm(-1), via (34)S and (15)NO isotope shifts. The electronic and vibrational properties of the S = 32 (FeNO)(7) unit are rationalized in terms of a limiting formulation involving a high-spin (S = 52) Fe(3+) center antiferromagnetically coupled to a (S = 1) NO(-) anion, with a highly covalent Fe(3+)-NO(-) interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.

A hyperthermophilic plant-type [2Fe-2S] ferredoxin from Aquifex aeolicus is stabilized by a disulfide bond.

A [2Fe-2S] ferredoxin (Fd1) from the hyperthermophilic bacterium Aquifex aeolicus has been obtained by heterologous expression of the encoding gene in Escherichia coli. Sequence comparisons show that this protein belongs to the extended family of plant- and mammalian-type [2Fe-2S] ferredoxins but also indicate that it is not closely similar to either the plant-type or mammalian-type subfamilies. Instead, it appears to bear some similarity to novel members of this family, in particular the Isc-type ferredoxins involved in the assembly of iron-sulfur clusters in vivo. The two redox levels of the [2Fe-2S](2+/+) metal site of A. aeolicus ferredoxin have been studied by UV-visible, resonance Raman, EPR, variable temperature magnetic circular dichroism, and Mössbauer spectroscopies. A full-spin Hamiltonian analysis is given for the Mössbauer spectra. In aggregate, the spectroscopic data reveal differences with both the plant-type and mammalian-type ferredoxins, in keeping with the sequence comparisons. The midpoint potential of the [2Fe-2S](2+/+) couple, at -375 mV versus the normal hydrogen electrode, is more negative than those of mammalian-type ferredoxins and at the upper end of the range covered by plant-type ferredoxins. A. aeolicus ferredoxin contains two cysteines in addition to the four that are committed as ligands of the [2Fe-2S] cluster. These two residues have been shown by chemical modification and site-directed mutagenesis to form a disulfide bridge in the native protein. While that cystine unit plays a significant role in the exceptional thermostability of A. aeolicus ferredoxin (T(m) = 121 degrees C at pH 7 versus T(m) = 113 degrees C in a molecular variant where the disulfide bridge has been removed), it does not bear on the properties of the [2Fe-2S](2+/+) chromophore. This observation is consistent with the large distance (ca. 20 A) that is predicted to separate the iron-sulfur chromophore from the disulfide bridge.

Spectroscopic investigation of the nickel-containing porphinoid cofactor F(430). Comparison of the free cofactor in the (+)1, (+)2 and (+)3 oxidation states with the cofactor bound to methyl-coenzyme M reductase in the silent, red and ox forms.

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F(430), a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F(430) in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F(430), variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F(430)M) in the (+)1, (+)2 and (+)3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle pi orbitals that are unique to Ni(I) forms of F(430). Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d-d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F(430) macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1.

Spectroscopic studies of Pyrococcus furiosus superoxide reductase: implications for active-site structures and the catalytic mechanism.

The combination of UV/visible/NIR absorption, CD and variable-temperature magnetic circular dichroism (VTMCD), EPR, and X-ray absorption (XAS) spectroscopies has been used to investigate the electronic and structural properties of the oxidized and reduced forms of Pyrococcus furiosus superoxide reductase (SOR) as a function of pH and exogenous ligand binding. XAS shows that the mononuclear ferric center in the oxidized enzyme is very susceptible to photoreduction in the X-ray beam. This observation facilitates interpretation of ground- and excited-state electronic properties and the EXAFS results for the oxidized enzyme in terms of the published X-ray crystallographic data (Yeh, A. P.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508). In the oxidized state, the mononuclear ferric active site has octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands. Fe EXAFS are best fit by one Fe-S at 2.36 A and five Fe-N/O at an average distance of 2.12 A. The EPR-determined spin Hamiltonian parameters for the high-spin (S = (5)/(2)) ferric site in the resting enzyme, D = -0.50 +/- 0.05 cm(-1) and E/D = 0.06, are consistent with tetragonally compressed octahedral coordination geometry. UV/visible absorption and VTMCD studies facilitate resolution and assignment of pi His --> Fe(3+)(t(2g)) and (Cys)S(p) --> Fe(3+)(t(2g)) charge-transfer transitions, and the polarizations deduced from MCD saturation magnetization studies indicate that the zero-field splitting (compression) axis corresponds to one of the axes with trans-histidyl ligands. EPR and VTMCD studies provide evidence of azide, ferrocyanide, hydroxide, and cyanide binding via displacement of the glutamate ligand. For azide, ferrocyanide, and hydroxide, ligand binding occurs with retention of the high-spin (S = 5/2) ground state (E/D = 0.27 and D < 0 for azide and ferrocyanide; E/D = 0.25 and D = +1.1 +/- 0.2 cm(-1) for hydroxide), whereas cyanide binding results in a low-spin (S = 1/2) species (g = 2.29, 2.25, 1.94). The ground-state and charge-transfer/ligand-field excited-state properties of the low-spin cyanide-bound derivative are shown to be consistent with a tetragonally elongated octahedral coordination with the elongation axis corresponding to an axis with trans-histidyl ligands. In the reduced state, the ferrous site of SOR is shown to have square-pyramidal coordination geometry in frozen solution with four equatorial histidines and one axial cysteine on the basis of XAS and UV and NIR VTMCD studies. Fe EXAFS are best fit by one Fe-S at 2.37 A and four Fe-N/O at an average distance of 2.15 A. VTMCD reveals a high-spin (S = 2) ferrous site with (Cys)S(p) --> Fe(2+) charge-transfer transitions in the UV region and (5)T(2g) --> (5)E(g) ligand-field transitions in the NIR region at 12400 and <5000 cm(-1). The ligand-field bands indicate square-pyramidal coordination geometry with 10Dq < 8700 cm(-1) and a large excited-state splitting, Delta (5)E(g) > 7400 cm(-1). Analysis of MCD saturation magnetization data leads to ground-state zero-field splitting parameters for the S = 2 ground state, D approximately +10 cm(-1) and E/D approximately 0.1, and complete assessment of ferrous d-orbital splitting. Azide binds weakly at the vacant coordination site of reduced SOR to give a coordination geometry intermediate between octahedral and square pyramidal with 10Dq = 9700 cm(-1) and Delta (5)E(g) = 4800 cm(-1). Cyanide binding results in an octahedral ferrous site with 10Dq = 10,900 cm(-1) and Delta (5)E(g) = 1750 cm(-1). The ability to bind exogenous ligands to both the ferrous and ferric sites of SOR is consistent with an inner-sphere catalytic mechanism involving superoxide binding at the ferrous site to yield a ferric-(hydro)peroxo intermediate. The structural and electronic properties of the SOR active site are discussed in relation to the role and bonding of the axial cysteine residue and the recent proposals for the catalytic mechanism.

Resonance Raman characterization of the mononuclear iron active-site vibrations and putative electron transport pathways in Pyrococcus furiosus superoxide reductase.

The resonance Raman spectrum of oxidized wild-type P. furiosus SOR at pH 7.5 and 10.5 has been investigated using excitation wavelengths between 406 and 676 nm, and vibrational modes have been assigned on the basis of isotope shifts resulting from global replacements of (32)S with (34)S, (14)N with (15)N, (56)Fe with (54)Fe, and exchange into a H(2)(18)O buffer. The results are interpreted in terms of the crystallographically defined active-site structure involving a six-coordinate mononuclear Fe center with four equatorial histidine ligands and axial cysteine and monodentate glutamate ligands (Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. C. (2000) Biochemistry 39, 2499-2508). Excitation into the intense (Cys)S(p(pi))-to-Fe(d(pi)) CT transition centered at 660 nm results in strong enhancement of modes at 298 cm(-1) and 323 cm(-1) that are assigned to extensively mixed cysteine S-C(beta)-C(alpha) bending and Fe-S(Cys) stretching modes, respectively. All other higher-energy vibrational modes are readily assigned to overtone or combination bands or to fundamentals corresponding to internal modes of the ligated cysteine. Weak enhancement of Fe-N(His) stretching modes is observed in the 200-250 cm(-1) region. The enhancement of internal cysteine modes and Fe-N(His) stretching modes are a consequence of a near-planar Fe-S-C(beta)-C(alpha)-N unit for the coordinated cysteine and significant (His)N(p(pi))-Fe(d(xy))-(Cys)S(p(pi)) orbital overlap, respectively, and have close parallels to type 1 copper proteins. By analogy with type 1 copper proteins, putative superexchange electron-transfer pathways to the mononuclear Fe active site are identified involving either the tyrosine and cysteine residues or the solvent-exposed deltaN histidine residue in a Y-C-X-X-H arrangement. Studies of wild-type at pH 10.5 and the E14A variant indicate that the resonance Raman spectrum is remarkably insensitive to changes in the ligand trans to cysteine and hence are inconclusive concerning the origin of the alkaline transition and the nature of sixth Fe ligand in the E14A variant.