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M W Adams

Publications and source records attributed to M W Adams.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization of an aminoacylase from the hyperthermophilic archaeon Pyrococcus furiosus.

Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-L-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 +/- 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl(2) (and to 74% by the addition of CoCl(2)). After reconstitution with ZnCl(2), the enzyme contained 2.85 +/- 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolar N-acylated L amino acids (Met, Ala, Val, and Leu), as well as N-formyl-L-methionine. The high K(m) values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity with N-acetyl-L-methionine as the substrate occurred at pH 6.5 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in the P. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7 lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn(2+) ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of the P. furiosus aminoacylase (> or = 50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

Amidohydrolases↗
PubMed2001

Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus.

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690+/-20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large ( approximately 1.6 MDa) complex that is not active. The apparent K(m) values and catalytic efficiencies for the substrates pyruvate and ATP (at 80 degrees C, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1). s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) x s(-1), respectively. Maximal activity was measured at pH 9.0 (at 80 degrees C) and at 90 degrees C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k(cat)/K(m) = 32 (mM. s(-1)]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration ( approximately 5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

Adenosine Monophosphate↗
PubMed2001

Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Culture Media↗
PubMed2001

DNA microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus: evidence for anNew type of sulfur-reducing enzyme complex.

DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S(primary)) on the levels of gene expression in cells grown at 95 degrees C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S(primary) and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S(primary). Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S(primary)-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S(primary)-grown cells, and these are also thought be involved in S(primary) reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S(primary)-grown cells, a finding consistent with the fact that growth on peptides is a S(primary)-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S(primary), although the reason for this is as yet unknown. Of the 20 S(primary)-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S(primary)-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.

Archaeal Proteins↗
PubMed2000

Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum. Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. Proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.

Iron-Sulfur Proteins↗
PubMed2000

Millisecond time scale conformational flexibility in a hyperthermophile protein at ambient temperature.

Rubredoxin from the hyperthermophile Pyrococcus furiosus is the most thermostable protein characterized to date with an estimated global unfolding rate of 10(-6) s(-1) at 100 degrees C. In marked contrast to these slow global dynamics, hydrogen exchange experiments here demonstrate that conformational opening for solvent access occurs in the approximately millisecond time frame or faster at 28 degrees C for all amide positions. Under these conditions all backbone amides with exchange protection factors between 10(4) and 10(6), for which EX(2) exchange kinetics were directly verified, have exchange activation energy values within 2-3 kcal/mol of that observed for unstructured peptides. The conformational flexibility of this protein is thus sufficient for water and base catalyst access to the exchanging amide with quite limited structural disruption. The common hypothesis that enhanced conformational rigidity in the folded native state underlies the increased thermal stability of hyperthermophile proteins is not supported by these data.

Archaeal Proteins↗
PubMed2000

Structures of the superoxide reductase from Pyrococcus furiosus in the oxidized and reduced states.

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

Crystallography, X-Ray↗
PubMed2000

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis.

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.

Aldehyde Oxidoreductases↗
PubMed2000

Organometallic iron: the key to biological hydrogen metabolism.

X-ray crystallography of iron-hydrogenases reveals that the active-site H-cluster contains an unmistakably organometallic dinuclear iron subcluster. The nickel-hydrogenases, which in general play different metabolic roles, have a distinct but related active-site structure. The new structural definition, combined with chemical analogs and theoretical treatment, points toward mechanistic understanding of iron-hydrogenases and the possibility of a unified mechanism for all hydrogenases.

Ferric Compounds↗
PubMed2000

Direct measurement of 1H-1H dipolar couplings in proteins: a complement to traditional NOE measurements.

An intensity-based constant-time COSY (CT-COSY) method is described for measuring 1H-1H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the 1H-1H distances are defined by secondary structure, such as 1H(alpha)-1H(N) and 1H(N)-1H(N) sequential distances in alpha-helices and beta-sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r3 distance dependence of directly observed dipolar couplings, as compared with the 1/r6 distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, 15N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules.

Acyl Carrier Protein↗
PubMed2000

Purification and characterization of a membrane-bound hydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 micromol of H(2) evolved/mg at 80 degrees C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (alpha and beta) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained approximately 1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H(2) as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the alpha and beta subunits were located in the genome database within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of P. furiosus and show that the alpha subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the alpha and beta subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospirillum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H(2) metabolism is coupled to energy conservation.

Catalysis↗
PubMed2000

Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction.

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M(r) of 320,000 +/- 20,000 and contains four different subunits with M(r)s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). The heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/- 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 +/- 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H(2) production with K(m) values near 70 microM and k(cat)/K(m) values near 350 min(-1) mM(-1). In contrast to hydrogenase I, hydrogenase II catalyzed the H(2)-dependent reduction of NAD (K(m), 128 microM; k(cat)/K(m), 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H(2) and NADPH served as electron donors for the reduction of elemental sulfur (S(0)) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H(2) using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) and one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nucleotide-binding sites in the gamma subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S(0) concentrations since it has a higher affinity than hydrogenase I for both S(0) and polysulfide.

Amino Acid Sequence↗
PubMed2000

Cloning, expression, and crystallization of Cpn60 proteins from Thermococcus litoralis.

Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli. Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms. The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.

Chaperonins↗
PubMed1999

Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus.

Glutamate dehydrogenase catalyses the oxidative deamination of glutamate to 2-oxoglutarate with concomitant reduction of NAD(P)(+), and has been shown to be widely distributed in nature across species ranging from psychrophiles to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus litoralis has been determined at 2.5 A resolution, and has been compared to that from the hyperthermophile Pyrococcus furiosus. The two enzymes are 87 % identical in sequence, yet differ 16-fold in their half-lives at 104 degrees C. This is the first reported comparative analysis of the structures of a multisubunit enzyme from two closely related yet distinct hyperthermophilies. The less stable T. litoralis enzyme has a decreased number of ion pair interactions; modified patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing efficiency; and substitutions which give rise to subtle but distinct shifts in both main-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structural studies.

Amino Acid Sequence↗
PubMed1999

Anaerobic microbes: oxygen detoxification without superoxide dismutase.

Superoxide reductase from the hyperthermophilic anaerobe Pyrococcus furiosus uses electrons from reduced nicotinamide adenine dinucleotide phosphate, by way of rubredoxin and an oxidoreductase, to reduce superoxide to hydrogen peroxide, which is then reduced to water by peroxidases. Unlike superoxide dismutase, the enzyme that protects aerobes from the toxic effects of oxygen, SOR does not catalyze the production of oxygen from superoxide and therefore confers a selective advantage on anaerobes. Superoxide reductase and associated proteins are catalytically active 80 degrees C below the optimum growth temperature (100 degrees C) of P. furiosus, conditions under which the organism is likely to be exposed to oxygen.

Acetylation↗
PubMed1999

Stability and sulfur-reduction activity in non-aqueous phase liquids of the hydrogenase from the hyperthermophile Pyrococcus furiosus.

Hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus, catalyzes the reversible activation of H(2) gas and the reduction of elemental sulfur (S degrees ) at 90 degrees C and above. The pure enzyme, modified with polyethylene glycol (PEG), was soluble (> 5 mg/mL) in toluene and benzene with t(1/2) values of more than 6 h at 25 degrees C. At 100 degrees C the PEG-modified enzyme was less stable in aqueous solution (t(1/2) approximately 10 min) than the native (unmodified) enzyme (t(1/2) approximately 1 h), but they exhibited comparable H(2) evolution, H(2) oxidation, and S degrees reduction activities at 80 degrees C. The H(2) evolution activity of the modified enzyme was twice that of the unmodified enzyme at 25 degrees C. The PEG-modified enzyme did not catalyze S degrees reduction (at 80 degrees C) in pure toluene unless H(2)O was added. The mechanism by which hydrogenase produces H(2)S appears to involve H(2)O as the proton source and H(2) as the electron source. The inability of the modified hydrogenase to catalyze S degrees reduction in a homogeneous non-aqueous phase complicates potential applications of this enzyme.

Biotechnology↗
PubMed1999

Effects of mutations in aspartate 14 on the spectroscopic properties of the [Fe3S4]+,0 clusters in Pyrococcus furiosus ferredoxin.

The properties of [Fe(3)S(4)](+,0) clusters in wild-type and mutant forms of Pf Fd with Asp, Ser, Cys, Val, His, Asn, and Tyr residues occupying position 14, i.e., proximal to the three micro(2)-S atoms of the cluster, have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and resonance Raman (RR) spectroscopies. Two distinct types of [Fe(3)S(4)] clusters are identified on the basis of the breadth of the S = (1)/(2) [Fe(3)S(4)](+) EPR resonances and the marked differences in the VTMCD spectra of the S = 2 [Fe(3)S(4)](0) clusters. On the basis of the available NMR data for [Fe(3)S(4)](+, 0) clusters in ferredoxins, the distinctive properties of these two types of [Fe(3)S(4)] clusters are interpreted in terms of different locations of the more strongly coupled pair of irons in the oxidized clusters and the valence-delocalized pair in the reduced clusters. Near-IR VTMCD measurements indicate the presence of S = (9)/(2) valence-delocalized pairs in both types of [Fe(3)S(4)](0) clusters, and the spin-dependent delocalization energies associated with the Fe-Fe interactions were determined to be approximately 4300 cm(-)(1) in both cases. We conclude that the nature of the residue at position 14 in Pyrococcus furiosus ferredoxin is an important determinant of the location of the reducible pair of irons in a [Fe(3)S(4)](+,0) cluster, and the redox properties of the wild-type and mutant ferredoxins are discussed in light of these new results.

Aspartic Acid↗