SlyD is a protein involved in [NiFe]-hydrogenase enzyme maturation and, together with HypB and HypA proteins, contributes to the nickel delivery step. To understand the molecular details of this in vivo function, the nickel-binding activity of SlyD was investigated in vitro. SlyD is a monomeric protein that can chelate up to 7 nickel ions with an affinity in the sub-nanomolar range. By truncation and mutagenesis studies we show that the unique C-terminal metal-binding domain of this protein is required for Ni(II) binding and that the protein coordinates this metal non-cooperatively. This activity of SlyD supports the proposed in vivo role of SlyD in nickel homeostasis. In addition to nickel, SlyD can bind a variety of other types of transition metals. Therefore it was feasible that the protein contributes to homeostasis of metals other than nickel. To test this hypothesis, the metal selectivity of the protein was examined. The preference of SlyD for the metals examined could be ordered as follows, Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) << Cu(I) indicating that the affinity of SlyD for the different metals follows the Irving-Williams series of metal-complex stabilities. Although the protein is unable to overcome the large thermodynamic preference in vitro for Cu(I) and exclude Zn(II) chelation, in vivo studies suggest a Ni(II)-specific function for the protein. To understand the function of SlyD as a metallochaperone, its interaction with HypB was investigated. This investigation revealed that SlyD plays a role in Ni(II) storage in E. coli and can function as a Ni(II)-donor to HypB. This study also revealed that SlyD can modulate the metal-binding as well as the GTPase activities of HypB. Based on the experimental data, a role for the HypB-SlyD complex in [NiFe]-hydrogenase biosynthesis is presented.

Accessory hydrogen uptake genes have been identified in a region of the Azotobacter chroococcum genome about 5 kb downstream of the hydrogenase structural genes (hupSL). DNA sequencing has revealed six genes (hupABYCDE) in this region. These genes are probably transcribed in the same direction as hupSL but are probably in a different operon. Mutational analysis had shown that disruption of the hupB, hupY, hupD and hupE genes gives a Hup$ sp-$ phenotype. In the present work additional mutational analysis, using Tn5, a Tn5 -derivative containing a promoterless lacZ gene, and a kanamycin resistance gene, confirms the direction of transcription and the separate nature of the hupABYCDE operon, and extends the region known to be necessary for Hup activity to hupA and possibly to 1.6 kb upstream of hupA.

University, Diss., 2005--Düsseldorf.

abstract: Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and catalyze hydrogen oxidation in vivo. In contrast, the group 3 [NiFe]-hydrogenases are heteromultimeric, bifunctional enzymes that fulfill various cellular roles. In this dissertation, protein film electrochemistry (PFE) is used to characterize the catalytic properties of two group 3 [NiFe]-hydrogenases: HoxEFUYH from Synechocystsis sp. PCC 6803 and SHI from Pyrococcus furiosus. First, HoxEFUYH is shown to be biased towards hydrogen production. Upon exposure to oxygen, HoxEFUYH inactivates to two states, both of which can be reactivated on the timescale of seconds. Second, we show that PfSHI is the first example of an oxygen tolerant [NiFe]-hydrogenase that produces two inactive states upon exposure to oxygen. Both inactive states are analogous to those characterized for HoxEFUYH, but oxygen exposed PfSHI produces a greater fraction that reactivates at high potentials, enabling hydrogen oxidation in the presence of oxygen. Third, it is shown that removing the NAD(P)-reducing subunits from PfSHI leads to a decrease in bias towards hydrogen oxidation and renders the enzyme oxygen sensitive. Both traits are likely due to impaired intramolecular electron transfer. Mechanistic hypotheseses for these functional differences are considered. / Dissertation/Thesis / Ph.D. Biochemistry 2012

abstract: The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

abstract: There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo. / Dissertation/Thesis / Ph.D. Biochemistry 2013

Using proteolytic probes, the pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was analyzed for its native topography in the cytoplasmic membrane. Before analyses could be performed, the isolation of transhydrogenase-enriched ISO (inside-out) cytoplasmic membrane vesicles was accomplished by modification of the procedure followed by Clarke (Clarke, D. M. and Bragg, P. D. (1985) Eur. J. Biochem. 149, 517-523) in purifying the enzyme from overexpressing E.coli JM83pDC21 cells. Two major changes were made. One was that the solubilization of the bacterial membrane and subsequent purification steps were omitted. The other was the separation of outer membranes from the cytoplasmic membrane preparation by sucrose gradient density centrifugation. This was essential owing to the contaminating presence of a 30 kD protein in the outer membrane of the original preparation. Transhydrogenase-enriched RSO (right-side-out) membrane vesicles were isolated by a different procedure using lysozyme-mediated breakage of E.coli spheroplasts and subsequent vesicular reformation. To identify possible transhydrogenase fragments arising from proteolytic cleavage, anti-E.coli transhydrogenase polyclonal antibodies were generated in rabbits. Two sets of polyclonal antibodies were produced. One set cross-reacted with both the α (52 kD) and β (48 kD) subunits of the transhydrogenase. The other reacted with the α subunit only. Trypsin and proteinase K were the main proteolytic probes used against both ISO and RSO cytoplasmic membrane vesicles, although chymotrypsin was also used in preliminary experiments with ISO membrane vesicles. Identification of fragments resulting from proteolytic cleavage of the enzyme was obtained using anti-transhydrogenase antibodies and by N-terminal sequencing and/or C-terminal sequencing. In some of these experiments, isolation of the proteolytic fragments was necessary prior to analysis. This was done using a number of different methods. The particular methods applied, which included column chromatography strategies and elution procedures from SDS-Polyacrylamide gels, depended on the type of analysis carried out. The analyses indicated that the α subunit has at least a 41 kD sequence extending from its N-terminus which is exposed to the cytoplasmic side of the membrane. This sequence may contain an active site of the enzyme. This is suggested by the binding of this fragment to a NAD-affinity column. The membrane-imbedded region of the α subunit anchoring the 41 kD region predicted by hydropathy plotting (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) could not be detected by our methods. Susceptible tryptic cleavage sites along the 41 kD region were identified by partial proteolysis and may reflect areas in the subunit's tertiary or quaternary structure that are exposed to the surrounding medium. Major cleavage sites were at arg₁₅, Iys₂₂₇, Iys₂₆₄, arg₂₆₈, Iys₂₇₅, arg₃₅₅, and arg₃₆₁. There do not appear to be significant portions of the subunit protruding into the periplasm as neither trypsin nor proteinase K had any effect on the subunit in RSO-oriented membrane vesicles. Proteinase K experiments with ISO and RSO membrane vesicles suggest that a 20 kD portion of the β subunit is protected from cleavage and is imbedded in the membrane. The identity of this fragment could not be confirmed. Hydropathy analysis of the transhydrogenase gene-derived amino acid sequence (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) suggests that this could be a sequence extending from the N-terminus of the β subunit. This is a hydrophobic sequence containing 7 possible transmembranous helices and having a theoretical molecular weight in the range of 20 kD. The proteinase K results also indicate that the rest of the β subunit is exposed to the cytoplasmic side of themembrane rather than the periplasmic side. The results obtained here are consistent with hydropathy predictions made with regard to this subunit. In addition, two different experiments indicate that an α-α subunit interaction may be present in the oligomeric structure of the membrane-bound enzyme (Hou, C, Potier, M. and Bragg, P. D. (1990), Biochim. Biophys. Acta 1018, 61-66). Substrates of the enzyme did not appear to affect the transhydrogenase's general conformation upon binding as detected by experiments using partial tryptic proteolysis. Partial trypsinolysis also revealed that selective detergent extraction of transhydrogenase-enriched ISO vesicles with Triton X-100 and sodium cholate did not affect the overall conformation of the membrane-bound enzyme despite greatly reducing the enzymatic activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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No description available.

The coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme formed aggregates (1,000 kd) of a coenzyme F₄₂₀-active monomer (109 kd) composed of 1 each of a, β, and γ subunits (43.6, 36.7, xy and 28.8 kd, respectively). It contained 1 mol of FAD, 1 mol of nickel, 12-14 mols of iron, and 11 mols of acid-labile sulfide per mol of the 109 kd species, but no selenium. The amino acid sequence I---P--R-EGH-----EV was conserved in the N-terminus of a subunit of the enzyme and the largest subunits of nickel-containing hydrogenases from Methanobacterium thermoautotrophicum, Desulfovibrio baculatus, and Desulfovibrio gigag. FAD dissociated from the coenzyme F42O-reducing hydrogenase during reactivation with H2 and coenzyme F₄₂₀, unless KCl was present, yielding coenzyme F₄₂₀-inactive apoenzyme. The hydrogenase catalyzed H₂ production at a rate 3-fold less than that for H2 uptake. Specific antiserum inhibited the coenzyme F₄₂₀ dependent activity but not the methyl viologen-dependent activity of the purified enzyme. Cell extract of M. formicicum contained a coenzyme F₄₂₀-mediated formate hydrogenlyase system. Formate hydrogenlyase activity was reconstituted with coenzyme F₄₂₀-reducing hydrogenase, coenzyme F₄₂₀-reducing formate dehydrogenase, and coenzyme F₄₂₀, all purified from M. formicicum. The reconstituted system required FAD for maximal activity (kinetic Kd= 4 μM). without FAD, the formate dehydrogenase and hydrogenase rapidly lost coenzyme F₄₂₀-dependent activity relative to methyl viologen-dependent activity. Immunoadsorption of the formate dehydrogenase or hydrogenase from cell extract greatly reduced formate hydrogenlyase activity; addition of the purified enzymes restored activity. Formate hydrogenlyase activity of cell extract and the reconstituted system was reversible. The coenzyme F₄₂₀-reducing hydrogenase and formate dehydrogenase of M. formicicum were shown to be located at the cytoplasmic membrane using immunogold labeling of thin sectioned, Lowicryl-embedded cells. Neither enzyme was released from whole cells by osmotic shock treatment. / Ph. D.