Temperature-dependent iron motion in extremophile rubredoxins - no need for 'corresponding states'.

Extremophile organisms are known that can metabolize at temperatures down to - 25 °C (psychrophiles) and up to 122 °C (hyperthermophiles). Understanding viability under extreme conditions is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Information about the stability and dynamics of proteins under environmental extremes is an important factor in this regard. Here we compare the dynamics of small Fe-S proteins - rubredoxins - from psychrophilic and hyperthermophilic microorganisms, using three different nuclear techniques as well as molecular dynamics calculations to quantify motion at the Fe site. The theory of 'corresponding states' posits that homologous proteins from different extremophiles have comparable flexibilities at the optimum growth temperatures of their respective organisms. Although 'corresponding states' would predict greater flexibility for rubredoxins that operate at low temperatures, we find that from 4 to 300 K, the dynamics of the Fe sites in these homologous proteins are essentially equivalent.

Cluster-Dependent Charge-Transfer Dynamics in Iron-Sulfur Proteins.

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.

Synchrotron-based Nickel Mössbauer Spectroscopy.

We used a novel experimental setup to conduct the first synchrotron-based (61)Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. (61)NiCr2O4 was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional (61)Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, (61)Ni{N(SiMe3)Dipp}2, where Dipp = C6H3-2,6-(i)Pr2, was chosen as a sample with an "extreme" geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based (61)Ni Mössbauer spectroscopy, we examined the spectra of (61)Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et4N]2[(61)Ni(SPh)4] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based (61)Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources.

Identification of protein-bound dinitrosyl iron complexes by nuclear resonance vibrational spectroscopy.

We have applied (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N-Fe-N unit can be observed between 500 and 700 cm(-1) and are diagnostic indicators of the type of iron dinitrosyl species present. NRVS spectra for four iron dinitrosyl model compounds are presented and used as benchmarks for the identification of species formed in the reaction of Pyrococcus furiosus ferredoxin D14C with nitric oxide.

Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy--interpretation by molecular mechanics.

We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)(4) site in oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of approximately 255-275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes. The strongest ICV mode with 570 nm excitation is the symmetric Fe-S stretching mode near 310 cm(-1), compared to 313 cm(-1) in the low temperature resonance Raman. If the rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm(-1). Finally, there is a mode at approximately 500 cm(-1) which is similar to features near 508 cm(-1) in blue Cu proteins that have been attributed to excited state vibrations. Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in rubredoxins.

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.

Normal mode analysis of Pyrococcus furiosus rubredoxin via nuclear resonance vibrational spectroscopy (NRVS) and resonance raman spectroscopy.

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(S(cys))(4) site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also been investigated by resonance Raman spectroscopy. In the oxidized Rd NRVS, strong asymmetric Fe-S stretching modes are observed between 355 and 375 cm(-1); upon reduction these modes shift to 300-320 cm(-1). This is the first observation of Fe-S stretching modes in a reduced Rd. The peak in S-Fe-S bend mode intensity is at approximately 150 cm(-1) for the oxidized protein and only slightly lower in the reduced case. A third band occurs near 70 cm(-1) for both samples; this is assigned primarily as a collective motion of entire cysteine residues with respect to the central Fe. The (57)Fe partial vibrational density of states (PVDOS) were interpreted by normal mode analysis with optimization of Urey-Bradley force fields. The three main bands were qualitatively reproduced using a D(2)(d) Fe(SC)(4) model. A C(1) Fe(SCC)(4) model based on crystallographic coordinates was then used to simulate the splitting of the asymmetric stretching band into at least 3 components. Finally, a model employing complete cysteines and 2 additional neighboring atoms was used to reproduce the detailed structure of the PVDOS in the Fe-S stretch region. These results confirm the delocalization of the dynamic properties of the redox-active Fe site. Depending on the molecular model employed, the force constant K(Fe-S) for Fe-S stretching modes ranged from 1.24 to 1.32 mdyn/A. K(Fe-S) is clearly diminished in reduced Rd; values from approximately 0.89 to 1.00 mdyn/A were derived from different models. In contrast, in the final models the force constants for S-Fe-S bending motion, H(S-Fe-S), were 0.18 mdyn/A for oxidized Rd and 0.15 mdyn/A for reduced Rd. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.

Rubrerythrin from the hyperthermophilic archaeon Pyrococcus furiosus is a rubredoxin-dependent, iron-containing peroxidase.

Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 +/- 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85 degrees C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 micromol of rubredoxin oxidized/min/mg with apparent K(m) values of 35 and 70 microM for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 micromol of H(2)O(2) reduced/min/mg of rubrerythrin at 85 degrees C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide:rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

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.

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.

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.