An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)

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Analog hardware accelerators, which perform computation within a dense memory array, have the potential to overcome the major bottlenecks faced by digital hardware for data-heavy workloads such as deep learning. Exploiting the intrinsic computational advantages of memory arrays, however, has proven to be challenging principally due to the overhead imposed by the peripheral circuitry and due to the non-ideal properties of memory devices that play the role of the synapse. We review the existing implementations of these accelerators for deep supervised learning, organizing our discussion around the different levels of the accelerator design hierarchy, with an emphasis on circuits and architecture. We explore and consolidate the various approaches that have been proposed to address the critical challenges faced by analog accelerators, for both neural network inference and training, and highlight the key design trade-offs underlying these techniques.

Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 form predominates. The addition or removal of an electron to the appropriate form of this variant serves as a switch to a new molecular form of the cytochrome. Using the 2 x 2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms: kb,AC = 17 s-1 for Met80-Fe3+-His18 --> His82-Fe3+-His18 and kf,BD > 10 s-1 for His82-Fe2+-His18 --> Met80-Fe2+-His18; E0' = 247 mV for Met80-Fe3+/2+-His18 couple, E0' = 47 mV for His82-Fe3+/2+-His18 couple, and E0' = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18. Thermodynamic parameters, including the entropy of reaction, DeltaS0'Rxn, were determined for the net reduction/rearrangement reaction, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18, and compared to those for wild-type cytochrome, Met80-Fe3+-His18 + e- --> Met80-Fe2+-His18. For the Phe82His variant mixed redox couple, DeltaS0'Rxn = -80 J/mol.K compared to DeltaS0'Rxn = -52 J/mol.K for the wild-type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction.

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 'H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T , smaller than 25 ms.The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3Jm.H0, H-bond constraints and dihedral angle constraints on the Cys x2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine PCH, protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi uriciBiol. 243, 683-6951. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The Ca root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.Keywords: NMR ; solution structure ; paramagnetic metalloproteins ; ferredoxins ; iron-sulfur proteins.It has been recently shown that it is possible to obtain threedimensional structures of paramagnetic proteins in solution [ 1, 21 (Bertini, I., Eltis, D., Felli, I. C., Kastrau, D. H. W., Luchinat, C. and Piccioli, M., unpublished results). This can be achieved by combining classical NMR methods for structure determination [3-51 with tailored experiments to detect connectivities involving fast relaxing signals [6-101. It is known that the feasibility of a solution structure in the case of diamagnetic systems is dependent essentially on the molecular mass of the protein [1 11. In paramagnetic proteins, the feasibility crucially depends on the percentage of nuclei affected by paramagnetism.The solution structures of two 4Fe-4S high-potential ironsulfur proteins (Hipips), in both oxidation states, have been reCorrespondence...

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Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E 0 ‫؍‬ ؊233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c 3 group, which is supported by N-terminal sequence data up to the first heme binding site.The ability of Shewanella putrefaciens to grow with a variety of different electron acceptors including oxygen, nitrate, sulfur, thiosulfate, sulfite, fumarate, trimethylamine-N-oxide (TMAO), and metal ions (10, 11) raises questions about the architecture of the electron transport chain in this versatile organism. In particular, given the range of electron potentials covered by the various electron acceptors it utilizes, the question of which types and the numbers of cytochromes present becomes an interesting one. In this regard, little detailed work has been done on cytochromes from S. putrefaciens. Early work by Obuekwe and Westlake (12) with whole-cell spectra showed that cytochrome c-type cytochromes were present and that both the red cell coloration and cytochrome content were correlated with the presence of iron in the growth medium. Arnold et al. (2) also reported whole-cell spectra and concluded that cytochrome synthesis was induced by growth under conditions of low oxygen tension. Myers and Myers (9) studied the locations of cytochromes in S. putrefaciens, concluding that many c-type cytochromes were present and that they were primarily localized in the outer membrane. Morris et al. (7) resolved nine c-type cytochrome bands by DEAE-Sepharose chromatography. There were two principal components that eluted at 90 mM and 315 mM NaCl, both of which had low redox potentials. In all of the above-cited studies, cytochrome content and type were judged mainly from spectral data, using the intensity of the Soret band after treatment with dithionite, or by heme staining polyacrylamide gels with tetramethylbenzidine (18). In recent and more definitive studies, a large tetraheme flavocytochrome c fumarate reductase was purified from S. putrefaciens (NCIMB400), and the gene sequence was determined (6,13). This enzyme is a 63.8-kDa soluble protein, and unlike the usual membrane-bound fumarate reductase, it contains heme c instead of iron-sulfur centers, and the heme midpoint redox potentials of Ϫ220 and Ϫ320 mV are unusually low. In a recent study, Lies et al. (5) reported that S. putrefaciens produces isoprenoid quinones of three types, with menaquinones and isoprenoid types predominating under aerobic conditions and methylmenaquinone types being present under anaerobic growth conditions. In this study, we report the purificat...