Ineffective Orifice Area: Practical Limitations of Accurate EOA Assessment for Low-Gradient Heart Valve Prostheses.

PURPOSE: The goal of this study was to demonstrate the range in effective orifice area (EOA) values that may be possible given the ISO 5840 definition of EOA and the practical limits in the accurate measurement of pressure differential across large diameter valves. METHODS: A 31 mm mechanical valve was tested on a commercially available pulse duplicator configured for mitral valve testing and tuned to nominal conditions. The experimental data was used as a basis for performing Monte Carlo analyses with published specifications for commonly used pressure sensors as well as measurement equipment accuracy requirements described in ISO 5840. The sources of error were modeled as normally distributed random variables and the simulation was iterated 1,000,000 times. RESULTS: Experimentally-derived EOAs ranged from 2.7 to 5.0 cm2, while the Monte Carlo simulation provided results ranging from approximately 0.4 to 6.7 cm2. Many of these results are clearly non-physical with EOAs larger than the valve's geometric orifice area and exceedingly short positive pressure differential periods, yet they align with other published results for the same valve model. CONCLUSIONS: The volatility of the standard EOA formulation at low mean gradients combined with the difficulty in accurately measuring such small differentials with industry-standard fluid pressure transducers results in a performance metric which is very sensitive to test execution, particularly for low-gradient prostheses.

Directionality of electron-transfer reactions in photosystem I of prokaryotes: universality of the bidirectional electron-transfer model.

The electron-transfer (ET) reactions in photosystem I (PS I) of prokaryotes have been investigated in wild-type cells of the cyanobacterium Synechocystis sp. PCC 6803, and in two site-directed mutants in which the methionine residue of the reaction center subunits PsaA and PsaB, which acts as the axial ligand to the primary electron chlorophyll acceptor A(0), was substituted with histidine. Analysis by pulsed electron paramagnetic resonance spectroscopy at 100 K indicates the presence of two forms of the secondary spin-correlated radical pairs, which are assigned to [P(700)(+)A(1A)(-)] and [P(700)(+)A(1B)(-)], where A(1A) and A(1B) are the phylloquinone molecules bound to the PsaA and the PsaB reaction center subunits, respectively. Each of the secondary radical pair forms is selectively observed in either the PsaA-M688H or the PsaB-M668H mutant, whereas both radical pairs are observed in the wild type following reduction of the iron-sulfur cluster F(X), the intermediate electron acceptor between A(1) and the terminal acceptors F(A) and F(B). Analysis of the time and spectral dependence of the light-induced electron spin echo allows the resolution of structural differences between the [P(700)(+)A(1A)(-)] and [P(700)(+)A(1B)(-)] radical pairs. The interspin distance is 25.43 ± 0.01 Å for [P(700)(+)A(1A)(-)] and 24.25 ± 0.01 Å for [P(700)(+)A(1B)(-)]. Moreover, the relative orientation of the interspin vector is rotated by ~60° with respect to the g-tensor of the P(700)(+) radical. These estimates are in agreement with the crystallographic structural model, indicating that the cofactors bound to both reaction center subunits of prokaryotic PS I are actively involved in electron transport. This work supports the model that bidirectionality is a general property of type I reaction centers from both prokaryotes and eukaryotes, and contrasts with the situation for photosystem II and other type II reaction centers, in which ET is strongly asymmetric. A revised model that explains qualitatively the heterogeneity of ET reactions at cryogenic temperatures is discussed.

The modulus of fibroblast-populated collagen gels is not determined by final collagen and cell concentration: Experiments and an inclusion-based model.

The fibroblast-populated collagen lattice is an attractive model tissue for in vitro studies of cell behavior and as the basis for bioartificial tissues. In spite of its simplicity-containing only collagen and cells-the system is surprisingly difficult to describe mechanically because of the ability of the cells to remodel the matrix, including compaction at short times and synthesis and/or degradation (and cell proliferation) at longer times. The objectives of this work were to measure the equilibrium modulus of fibroblast-populated gels with different collagen and cell concentrations, and to use that characterization as the basis for a theoretical model that could be used to predict gel mechanics based on conditions. Although many observations were as expected (e.g., the gel compacts more when there are more cells in it, and the gel is stiffer when there is more collagen in it), an unexpected result arose: the final modulus of the gel was not dependent solely on the final composition. Even if it compacted more than a gel that was originally at a high collagen concentration, a gel that started at a low collagen concentration remained less stiff than the higher-concentration gel. In light of these results and experimental studies by others, we propose a model in which the gel compaction is not homogeneous but consists instead of extreme densification near the cells in an otherwise unchanged matrix. By treating the dense regions as spherical inclusions, we used classical composite material theory to develop an expression for the modulus of a compacted gel based on the initial collagen density and the final inclusion (i.e., cell) density. The new model fit the data for moderately compacted gels well but broke down, as expected, for larger volume fractions at which the underlying model assumptions did not apply.

Planar biaxial mechanical behavior of bioartificial tissues possessing prescribed fiber alignment.

Though it is widely accepted that fiber alignment has a great influence on the mechanical anisotropy of tissues, a systematic study of the influence of fiber alignment on the macroscopic mechanical behavior by native tissues is precluded due to their predefined microstructure and heterogeneity. Such a study is possible using collagen-based bioartificial tissues that allow for alignment to be prescribed during their fabrication. To generate a systemic variation of strength of fiber alignment, we made cruciform tissue constructs in Teflon molds that had arms of different aspect ratios. We implemented our anisotropic biphasic theory of tissue-equivalent mechanics to simulate the compaction by finite element analysis. Prior to tensile testing, the construct geometry was standardized by cutting test samples with a 1:1 cruciform punch after releasing constructs from the molds. Planar biaxial testing was performed on these samples, after stretching them to their in-mold dimensions to recover in-mold alignment, to observe the macroscopic mechanical response with simultaneous fiber alignment imaging using a polarimetry system. We found that the strength of fiber alignment of the samples prior to release from the molds linearly increased with anisotropy of the mold. In testing after release, modulus ratio (modulus in fiber direction/modulus in normal direction) was greater as the initial strength of fiber alignment increased, that is, as the aspect ratio increased. We also found that the fiber alignment strength and modulus ratio increased in a hyperbolic fashion with stretching for a sample of given aspect ratio.

Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal fibroblasts within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming growth factor beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and 20 mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.

Three-dimensional simulation of anisotropic cell-driven collagen gel compaction.

Tissue equivalents (TEs), formed by entrapping cells in a collagen gel, are an important model system for studying cell behavior. We have previously (Barocas and Tranquillo in J Biomech Eng 117:161-170, 1997a) developed an anisotropic biphasic theory of TE mechanics, which comprises five coupled partial differential equations describing interaction among cells and collagen fibers in the TE. The model equations, previously solved in one or two dimensions, were solved in three dimensions using an adaptive finite-element platform. The model was applied to three systems: a rectangular isometric cell traction assay, an otherwise- acellular gel containing two islands of cells, and an idealized tissue-engineered cardiac valve leaflet. In the first two cases, published experimental data were available for comparison, and the model results were consistent with the experimental observations. Fibers and cells aligned in the fixed direction in the isometric assay, and a region of strong fiber alignment arose between the two cell islands. For the valve problem, the alignment predicted by the model was generally similar to that observed experimentally, but an asymmetry in the experiment was not captured by the model.

An electron paramagnetic resonance investigation of the electron transfer reactions in the chlorophyll d containing photosystem I of Acaryochloris marina.

Electron paramagnetic resonance (EPR) spectroscopy reveals functional and structural similarities between the reaction centres of the chlorophyll d-binding photosystem I (PS I) and chlorophyll a-binding PS I. Continuous wave EPR spectrometry at 12K identifies iron-sulphur centres as terminal electron acceptors of chlorophyll d-binding PS I. A transient light-induced electron spin echo (ESE) signal indicates the presence of a quinone as the secondary electron acceptor (Q) between P(740)(+) and the iron-sulphur centres. The distance between P(740)(+) and Q(-) was estimated within point-dipole approximation as 25.23+/-0.05A, by the analysis of the electron spin echo envelope modulation.

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii.

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P(700) recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.

Bidirectional electron transfer in photosystem I: replacement of the symmetry-breaking tryptophan close to the PsaB-bound phylloquinone A1B with a glycine residue alters the redox properties of A1B and blocks forward electron transfer at cryogenic temperatures.

A conserved tryptophan residue located between the A(1B) and F(X) redox centres on the PsaB side of the Photosystem I reaction centre has been mutated to a glycine in Chlamydomonas reinhardtii, thereby matching the conserved residue found in the equivalent position on the PsaA side. This mutant (PsaB:W669G) was studied using EPR spectroscopy with a view to understanding the molecular basis of the reported kinetic differences in forward electron transfer from the A(1A) and the A(1B) phyllo(semi)quinones. The kinetics of A(1)(-) reoxidation due to forward electron transfer or charge recombination were measured by electron spin echo spectroscopy at 265 K and 100 K, respectively. At 265 K, the reoxidation kinetics are considerably lengthened in the mutant in comparison to the wild-type. Under conditions in which F(X) is initially oxidised the kinetics of charge recombination at 100 K are found to be biphasic in the mutant while they are substantially monophasic in the wild-type. Pre-reduction of F(X) leads to biphasic kinetics in the wild-type, but does not alter the already biphasic kinetic properties of the PsaB:W669G mutant. Reduction of the [4Fe-4S] clusters F(A) and F(B) by illumination at 15 K is suppressed in the mutant. The results provide further support for the bi-directional model of electron transfer in Photosystem I of C. reinhardtii, and indicate that the replacement of the tryptophan residue with glycine mainly affects the redox properties of the PsaB bound phylloquinone A(1B).

Biomechanical and microstructural characteristics of a collagen film-based corneal stroma equivalent.

The growth in refractive surgeries and corneal replacements has fueled interest in the development of a tissue-engineered cornea. This study characterizes the microstructure and biomechanical properties of film-based corneal stroma equivalents over time in culture. The increased collagen density in the films was hypothesized to result in improved mechanical properties both initially and over time. The microstructure of the film-based stromal equivalent was examined using atomic force microscopy and scanning electron microscopy; the mechanical properties, relaxed modulus, and ultimate tensile strength were quantified using uniaxial tensile testing. The dense, film-based stromal equivalent had a lamellae-like microstructure, which was notably different than the porous structure of sponges used previously. Seeded human corneal stromal fibroblasts remained on the surface of the film rather than migrating into the film and produced fibers of extracellular matrix with diameters of 35-75 nm. After an initial decrease during hydration, the relaxed modulus and ultimate tensile strength for fully hydrated collagen films were 0.4 +/- 0.2 MPa and 0.3 +/- 0.1 MPa, respectively. The mechanical properties of cell-seeded films mimicked those of control films. While further studies are needed to quantify the optical properties, the dense, lamellae-like structure of collagen films is a feasible scaffold for the development of tissue-engineered stroma.

Analysis of the spin-polarized electron spin echo of the [P700+ A1-] radical pair of photosystem I indicates that both reaction center subunits are competent in electron transfer in cyanobacteria, green algae, and higher plants.

The decay of the light-induced spin-correlated radical pair [P700+ A1-] and the associated electron spin echo envelope modulation (ESEEM) have been studied in either thylakoid membranes, cellular membranes, or purified photosystem I prepared from the wild-type strains of Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, and Spinaceae oleracea. The decay of the spin-correlated radical pair is described in the wild-type membrane by two exponential components with lifetimes of 2-4 and 16-25 micros. The proportions of the two components can be altered by preillumination of the membranes in the presence of reductant at temperatures lower than 220 K, which leads to the complete reduction of the iron-sulfur electron acceptors F(A), F(B), and F(X) and partial photoaccumulation of the reduced quinone electron acceptor A1A-. The "out-of-phase" (OOP) ESEEM attributed to the [P700+ A1-] radical pair has been investigated in the three species as a function of the preillumination treatment. Values of the dipolar (D) and the exchange (J) interactions were extracted by time-domain fitting of the OOP-ESEEM. The results obtained in the wild-type systems are compared with two site-directed mutants of C. reinhardtii [Santabarbara et al. (2005) Biochemistry 44, 2119-2128], in which the spin-polarized signal on either the PsaA- or PsaB-bound electron transfer pathway is suppressed so that the radical pair formed on each electron transfer branch could be monitored selectively. This comparison indicates that when all of the iron-sulfur centers are oxidized, only the echo modulation associated with the A branch [P700+ A1A-] radical pair is observed. The reduction of the iron-sulfur clusters and the quinone A1 by preillumination treatment induces a shift in the ESEEM frequency. In all of the systems investigated this observation can be interpreted in terms of different proportions of the signal associated with the [P700+ A1A-] and [P700+ A1B-] radical pairs, suggesting that bidirectionality of electron transfer in photosystem I is a common feature of all species rather than being confined to green algae.

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X).

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).

Ammonia displaces methanol bound to the water oxidizing complex of photosystem II in the S2 state.

Ammonia and methanol both bind to the water oxidising complex of photosystem II during its turnover, possibly at sites where water binds during the normal water oxidation process. We have investigated the interaction between these two water analogues at the S2 state of the water oxidising cycle using electron magnetic resonance techniques. We find evidence that ammonia displaces methanol from its binding site.

Photosynthetic water oxidation: the role of tyrosine radicals.

This mini-review outlines the involvement of the tyrosine electron carriers, Y(D) and Y(Z), in the mechanism of electron transfer from water to P680. We discuss our data and put forward our ideas on the role of Y(D) and Y(Z).

Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas.

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.

Effect of hydroxylamine on photosystem II: reinvestigation of electron paramagnetic resonance characteristics reveals possible S state intermediates.

Previous work in many laboratories has established that hydroxylamine reduces the S(1) state of the water oxidizing complex (WOC) in one-electron steps. Significant levels of what can now be defined as the S(-1)* state are achieved by specific (concentration and incubation length) hydroxylamine treatments. This state has already been studied by electron paramagnetic resonance spectrometry (EPR), and unusual EPR signals were noted (for example, see Sivaraja, M., and Dismukes, G. C. (1988) Biochemistry 27, 3467-3475). We have now reinvestigated these initial experiments and confirmed many of the original observations. We then utilized more recent EPR markers for the S(0) and S(1) states to further explore the S(-1)* state. The broad radical "split" type EPR signal, produced by 200 K illumination of samples prepared to give a high yield of the S(-1)* state, is shown to most likely reflect a trapped intermediate state between S(-1)* and S(0)*, since samples where this signal is present can be warmed in the dark to produce S(0)*. The threshold for advancement from S(-1)* to S(0)* is near 200 K, as the yield of broad radical decreases and S(0)* multiline EPR signal increases with length of 200 K illumination. Advancement of S(0)* to S(1) is limited at 200 K, but S(1) can be restored by 273 K illumination. Illumination of these hydroxylamine-treated samples at temperatures below 77 K gives a second broad radical EPR signal. The line shape, decay, and other properties of this new radical signal suggest that it may arise from an interaction in the S(-2)* or lower S states, which are probably present in low yield in these samples. Illumination below 20 K of S(0)* state samples containing methanol, and therefore exhibiting the S(0) multiline signal, gives rise to a third broad radical with distinctive line shape. The characteristics of the three broad radicals are similar to those found from interactions between Y(Z)(*) and other S states. The evidence is presented that they do represent intermediate states in S state turnover. Further work is now needed to identify these radicals.

Stabilization of iron-sulfur cluster F(X) by intra-subunit interactions unraveled by suppressor and second site-directed mutations in PsaB of Photosystem I.

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.

Photoaccumulation of the PsaB phyllosemiquinone in photosystem I of Chlamydomonas reinhardtii.

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700(z.rad;+). Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700(z.rad;+), and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.

An iron-sulfur cluster in the family 4 uracil-DNA glycosylases.

The 25-kDa Family 4 uracil-DNA glycosylase (UDG) from Pyrobaculum aerophilum has been expressed and purified in large quantities for structural analysis. In the process we observed it to be colored and subsequently found that it contained iron. Here we demonstrate that P. aerophilum UDG has an iron-sulfur center with the EPR characteristics typical of a 4Fe4S high potential iron protein. Interestingly, it does not share any sequence similarity with the classic iron-sulfur proteins, although four cysteines (which are strongly conserved in the thermophilic members of Family 4 UDGs) may represent the metal coordinating residues. The conservation of these residues in other members of the family suggest that 4Fe4S clusters are a common feature. Although 4Fe4S clusters have been observed previously in Nth/MutY DNA repair enzymes, this is the first observation of such a feature in the UDG structural superfamily. Similar to the Nth/MutY enzymes, the Family 4 UDG centers probably play a structural rather than a catalytic role.