Overexpression, isolation, and spectroscopic characterization of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC 6803.
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 (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S](+) and at least one [2Fe2S](+) cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.
Germer
Frauke
F
Botanisches Institut, Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany.
Zebger
Ingo
I
Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17, Juni 135, D-10623 Berlin, Germany.
Saggu
Miguel
M
Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17, Juni 135, D-10623 Berlin, Germany.
Lendzian
Friedhelm
F
Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17, Juni 135, D-10623 Berlin, Germany.
Schulz
Rüdiger
R
Botanisches Institut, Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany.
Appel
Jens
J
School of Life Sciences, Arizona State University, Tempe, Arizona 85287. Electronic address: jens.appel@asu.edu.
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D015870:Gene Expression; D006864:Hydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007501:Iron / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D046937:Nostoc / Q000201:enzymology / Q000235:genetics; D010084:Oxidation-Reduction; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D009249:NADP; D009532:Nickel; D007501:Iron; D006864:Hydrogenase