Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

Abstract

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

“…2). Using this approach, we were able to identify a complex containing all five Hox subunits, again consistent with the composition of the purified pentameric complex reported in the literature (19,20). The apparent molecular mass of this complex is ϳ160 kDa, close to the calculated mass based on the predicted sizes of the individual subunits (175 kDa).…”

“…Other purifications in R. eutropha utilizing gene knockouts and ectopic expression demonstrate that HoxYH and HoxFU subcomplexes can be isolated, although these subcomplexes were reportedly unstable (13). In A. vinosum (12) and the cyanobacterium Gleocapsa alpicola CALU 743 (17), attempted purifications of native complex resulted in isolation of only HoxYH, whereas in T. roseopersicina (18) and Synechocystis (19,20), intact HoxEFUYH complexes were purified. Purification of the Synechocystis Hox hydrogenase by Schmitz et al (19) resulted in a dimer of a 1:1:1:1:1 HoxE/F/U/Y/H complex via a series of column chromatography steps, whereas Germer et al (20) purified a complex with a 0.2:2:2:1:1 HoxE/F/U/Y/H ratio using a StrepIItagged HoxF, raising questions about Hox complex assembly and association in vivo.…”

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic, Nitrate-limiting Conditions

“…Hydrogenase Purification-Hydrogenase was purified as described (19). Briefly, a mutant containing an N-terminal Strep tag fused to HoxF was harvested.…”

“…The hydrogenase of Synechocystis was purified as already described (19) and subjected to different electron donors. Hydrogen was only produced upon addition of dithionite-reduced MV, whereas all natural electron carriers as NADH, NADPH, reduced flavodoxin, and reduced ferredoxin failed.…”

“…Remarkably, the hydrogenase part alone has H 2 evolving activity with reduced MV (15,18). Analyses concerning the stoichiometry of the purified hydrogenase in our laboratory revealed only substoichiometic levels of HoxE (19). HoxE deletion mutants are devoid of hydrogen production in vivo, and the enzyme no longer evolves hydrogen upon addition of NAD(P)H in cell-free extracts (13).…”