William R. Dunham

On the structure of the iron-sulfur complex in the two-iron ferredoxins

Abstract Recent spectroscopic and magnetic susceptibility studies of the iron center in the two-iron ferredoxins provide criteria which any model for the iron-sulfur complex in these proteins must satisfy. These criteria are most stringent for parsley and spinach ferredoxin: the reduced proteins contain a high-spin ferric atom antiferromagnetically exchange-coupled (presumably via sulfide bridging ligands) to a high-spin ferrous atom. In the oxidized proteins the iron atoms are antiferromagnetically spin-coupled, high-spin ferric atoms. Arguments are given to substantiate the claim that the ferrous atom in the reduced protein is ligated by four sulfur atoms in a distorted tetrahedral configuration: two are the bridging sulfides, two are cysteinyl sulfurs. A treatment of proton contact shifts based upon the above model is pertinent to proton magnetic resonance data already available and provides a means to identify directly the ligands at both iron atoms via further PMR experiments.

The magnetic susceptibility of spinach ferredoxin from 77–250°K: A measurement of the antiferromagnetic coupling between the two iron atoms☆

Abstract The magnetic susceptibility of oxidized and reduced spinach ferredoxin has been measured over the temperature range 77–250°K. Anomalous behavior is observed in both oxidation states and the data can be interpreted by assuming an exchange interaction between the metal ions. The exchange constant is estimated to be 183 cm −1 in oxidized ferredoxin and ≤ 100 cm −1 in reduced ferredoxin.

A Statistical Theory for Powder EPR in Distributed Systems

p, whose principal elements are random variables. The p and g tensors are not necessarily colinear. The observed EPR linewidth results from a distribution in the effective g value as a function of (a) the joint distribution function of the elements of the p tensor and (b) the spatial relationship between the two principal axis systems involved. The theory is reformulated in terms of matrices that facilitate a direct comparison with earlier work. Two previous theories of g strain represent different subsets of the general theory, namely, the case of zero rotation between axis systems and the case with nonzero rotation and full correlation between elements of the p

A novel S=3/2 EPR signal associated with native Fe-proteins of nitrogenase

In addition to their g = 1.94 EPR signal, nitrogenase Fe‐proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g ≅5. Temperature dependence of the signal was consistent with an S = system with negative zero‐field splitting, d = −5 ± 0.7 cm−1. The m s, = ± ground state doublet gives rise to a transition with g eff = 5.90 and the transition within the excited m s = ± doublet has a split g eff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin mol−1 which summed with the spin intensity of the S = g = 1.94 line to roughly 1 . MgATP and MgADP decreased the intensity of the s = signal with no concomitant changes in intensity of the s = signal.

Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.

Coagulation factor VIII (FVIII) is a heterodimer consisting of a light chain of 80 kDa (domains A3-C1-C2) in a metal ion-dependent association with a 220-kDa heavy chain (domains A1-A2-B). The nature of the metal ion-dependent association between the heavy and light chains was investigated using atomic absorption spectroscopy, electron paramagnetic resonance spectroscopy (EPR), and site-directed mutagenesis and expression of the FVIII cDNA. Whereas copper ion was not detected in intact recombinant FVIII, EDTA dissociation of the chains yielded an EPR signal consistent with 1 mol of Cu(I)/mol of active protein, supporting the hypothesis that a single molecule of reduced copper ion is buried within intact FVIII and is released and oxidized upon treatment with EDTA. Cu(I), and not Cu(II), was able to reconstitute FVIII activity from dissociated chains, demonstrating a requirement for Cu(I) in FVIII function. Three potential copper ion binding sites exist within FVIII: one type-2 site and two type-1 sites. The importance of these potential copper ion ligands was tested by studying the effect of site-directed mutants. Of the two histidines that compose the type-2 binding site, the His-1957 → Ala mutant displayed secretion, light and heavy chain assembly, and activity similar to wild-type FVIII, while mutant His-99 → Ala was partially defective for secretion and had low levels of heavy and light chain association and activity. In contrast, FVIII having the mutation Cys-310 → Ser within the type-1 copper binding site in the A1 domain was inactive and partially defective for secretion from the cell, and the heavy and light chains of the secreted protein were not associated. Mutant Cys-2000 → Ser within the A3 domain displayed secretion, assembly, and activity similar to that for wild-type FVIII. These results support the hypothesis that Cu(I) is buried within the type-1 copper binding site within the A1 domain and is required for FVIII chain association and activity.

Quantitative Numerical Analysis of g Strain in the EPR of Distributed Systems and Its Importance for Multicenter Metalloproteins

A method for simulation of inhomogeneously broadened EPR of metallo-proteins based on recent theoretical advances is surveyed critically in terms of efficiency and accuracy. From the quality of the experimental spectrum, minimal boundary conditions are established for the spatial integration over the g-strained polycrystal. Computational efficiency is achieved by generating the S = 12 spectrum as an absorption in g space, reducing the number of molecular orientations computed by filtering mosaic artifacts from the Fourier-transformed spectrum, and generating the lineshape due to g strain from a tabulated distribution function. These techniques provide a reduction in computation time by some two orders of magnitude and make the data analysis of EPR of metalloproteins by minimization practical. The resulting simulation program is superior to current approaches in that it does not introduce artifactual multiplicities, and it is expected to require a smaller number of fitting parameters for the quantitative analysis of most cases. To illustrate its potential, the method is applied to EPR data from the iron-sulfur centers in NADH:Q oxidoreductase and in QH2:ferricytochrome c oxidoreductase, clarifying existing controversies on the stoichiometries of these centers.

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.

EPR of a novel high-spin component in activated hydrogenase from Desulfovibrio vulgaris (Hildenborough)

The EPR of reoxidized hydrogenase from Desulfovibrio vulgaris (H.) has been reinvestigated. In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728‐3732] we find the axial signal with g = 2.06; 2.01 to be only a minor component of concentration 0.03 . In the spectrum of fully active reoxidized enzyme this signal is overshadowed by a rhombic signal (0.1 ) with g = 2.11; 2.05; 2.00 reminiscent of the only signal found for other oxidized bidirectional hydrogenases. In addition, a novel signal has been detected with g eff = 5.0 which, under the assumptions that S = 2 and ¦Δm s¦= 2, quantitates to roughly one . Ethylene glycol affects the relative intensity of the different signals. It is suggested that O2 sensitization parallels a spin‐state transition of an iron‐sulfur cluster.

Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferrodoxin. Possible new fingerprints for three iron clusters

We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus. First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers. This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2. Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the [4Fe-4S]+ (S = 1/2) and the reduced 3Fe center. A theoretical explanation is given for the observation of interaction signals with constant effective g values.

An Analysis of g Strain in the EPR of Two (2Fe-2S) Ferredoxins. Evidence for a Protein Rigidity Model

Abstract Replacing current notions of a paramagnetic center in a metalloprotein as a single entity in vivo with the more realistic concept of an ensemble of spin systems, each uniquely disturbed by its own surrounding protein, leads to a rigorous description of the spectroscopic factor, g, as a random variable whose statistical properties contain information on the rigidity of the protein. Generation of a consistent set of accurate simulations of very low-noise, multifrvquency (3, 9, 15 GHz) EPR data from selected S = 1 2 proteins has now been achieved. This consistency lends support to the physical and biological inferences drawn from such simulations. The spectral contribution of magnetic hyperfine line-broadening is minimized by studying the 56Fe reconstituted [2Fe2S] cluster in fully deuterated ferredoxin from Synechococcus lividus and the 2H2O exchanged [2Fe2S] ferredoxin from Pseudomonas putida. High-resolution Mossbauer data on oxidized and reduced 57Fe reconstituted S. lividus ferredoxin are also presented. The oxidized spectrum shows that the inequivalence of the two ferric ions in a [2Fe2S] cluster can be resolved as two Mossbauer lines. The complete absence of this splitting in the ferric fines of the reduced spectrum is definitive proof that the reducing electron always resides at the same 56Fe atom in frozen aqueous solutions. To explain the distributed nature of the paramagnetic site in the ferredoxins, three models are considered: (1) a multiplicity of EPR states; (2) external perturbations to the molecular Hamiltonian; (3) a distribution in the crystal field Hamiltonian parameters. The first model is discarded, the second is possible but difficult to verify, and the third model is shown to fit the data well. The latter comparison requires a correction to literature expressions for the g and A tensors in [2Fe2S] clusters. Statistical analysis strongly suggests that the EPR of metalloproteins in its details is a reflection of protein structure that distributes its spatial coordinates, accommodating different levels of rigidity, the more flexible parts being located at the outside.