Light and Dark Reactions of the Uptake Hydrogenase in Anabaena 7120

Abstract

Reactions of the uptake hydrogenase from Anabaena 7120 (A.T.C.C. 27893, Nostoc muscorum) were examined in whole filaments, isolated heterocysts, and membrane particles. Whole ifiaments or isolated heterocysts that contained nitrogenase consumed H2 in the presence of C2H2 or N2 in a light-dependent reaction. If nitrogenase was inactivated by O2 shock, filaments catalyzed H2 uptake to an unidentified endogenous acceptor in the light. Addition of N03-or N02-enhanced these rates. Isolated heterocysts consumed H2 in the dark in the presence of electron acceptors with positive midpoint potentials, and these reactions were not enhanced by light. With acceptors of negative midpoint potential, significant light enhancement of H2 uptake occurred. Maximum rates of light-dependent uptake were approximately 25% of the maximum dark rates observed. Membrane particles prepared from isolated heterocysts showed similar specificity for electron acceptors. These particles catalyzed a cyanidesensitive oxyhydrogen reaction that was inactivated by 02 at 02 concentrations above 2%. Light-dependent H2 uptake to low potential acceptors by these particles was inhibited by dibromothymoquinone but was insensitive to cyanide. In the presence of 02, light-dependent H2 uptake occurred sunultaneously with the oxyhydrogen reaction. The pH optima for both types of H2 uptake were near 7.0. These results further clarify the role of uptake hydrogenase in donating electrons to both the photosynthetic and respiratory electron transport chains of Anabaena.Uptake hydrogenases have been found in several aerobic N2-fixing organisms (1,6,12), and these enzymes share several common properties. They are membrane-bound (7,12,28), couple to 02 consumption via a Cyt-containing electron transport system, and through this reaction provide a source of ATP (7, 9, 21). Several authors have suggested that these uptake hydrogenases function to recycle the H2 evolved by nitrogenase (4,7,9,21).In blue-green algae, membrane-bound hydrogenases can couple to two different electron transport pathways. Peschek has demonstrated respiratory H2 uptake and photosynthetic H2 uptake in Anacystis nidulans (19,20) in which the hydrogenase was induced by growth under a gas phase containing H2. In the dark, both H2 uptake systems could react only with acceptors having a positive midpoint potential; however, upon illumination acceptors with a negative potential could also be reduced.

Cloning and sequence analysis of the gene encoding the low potential cytochrome c of Synechocystis PCC 6803

“…The bound form of the cytochrome may be a constitutively expressed component of the photosynthetic apparatus and the soluble cytochrome may, in some species, be induced by anaerobic conditions just as hydrogenase is induced. The soluble cytochrome may function in dark, anaerobic fermentation [22] and the membranebound cytochrome may function in light driven uptake of hydrogen [23,24].…”

Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans

“…The two open reading frames ORF8 and ORF3 between the structural genes hoxFUYH in A. variabilis [7] are unusual for hydrogenase gene clusters. The function, if any, of the proteins encoded by these ORFs in the expression or regulation of this bidirectional hydrogenase is likely not correlated with heterocyst differentiation and/or nitrogenase synthesis, since the enzyme also occurs in vegetative cells [29]. Both expression and activity of the bidirectional hydrogenase in cyanobacteria are subject to complex and not yet fully understood regulation [1,2,30].…”

“…It occurs in both heterocysts and vegetative cells of Anabaena sp. [3,4] as well as in the unicellular A. nidulans [5]. Immunogold labeling experiments [5] as well as results with spheroplasts [6] indicated its location at the cytoplasmic membrane of A. nidulans.…”

Concentration and Function of Membrane-Bound Cytochromes in Cyanobacterial Heterocysts

“…As an electronic analog of a semiquinone, HQNO appears to exert potent inhibition in situations where one-electron carriers such as Cyt must react with quinones carrying two electrons, with a semiquinone as a likely intermediate. HQNO has been shown previously to block H2 uptake with a number ofdifferentelectron acceptors (13). The sites of inhibiton relevant to the H2 uptake pathway of heterocyst membranes are shown in Figure 4 Figure 5.…”

“…H2 uptake was measured amperometrically as in Houchins and Burris (13) except that the buffer was 40 mM Hepes, pH 7.5. Heterocyst membranes used for measurement of H2 uptake rates and hydrogenase-dependent difference spectra ( …”