4.6 Article

The structure and reactivity of the HoxEFU complex from the cyanobacterium Synechocystis sp. PCC 6803

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 28, Pages 9445-9454

Publisher

ELSEVIER
DOI: 10.1074/jbc.RA120.013136

Keywords

Synechocystis; HoxEFU; diaphorase; photosynthesis; kinetics; protein cross-linking; electron paramagnetic resonance (EPR); bidirectional hydrogenase; protein-protein interaction; nickel-iron enzyme; cooperativity; hydrogenase; nickel

Funding

  1. U.S. Department of Energy Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, Photosynthetic Systems Program

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Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H-2)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation. Moreover, outstanding questions about the reactivity of Hox with natural substrates and the site(s) of substrate interactions and coupling of H-2, NAD(P)H, and Fdx remain to be resolved. To address these questions, here we analyzed recombinantly produced HoxEFU by electron paramagnetic resonance spectroscopy and kinetic assays with natural substrates. The purified HoxEFU subcomplex catalyzed electron transfer reactions among NAD(P)H, flavodoxin, and several ferredoxins, thus functioningin vitroas a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD(+)and NADP(+)were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)(+), Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD(+). MS-based mapping identified an Fdx1-binding site at the junction of HoxE and HoxF, adjacent to iron-sulfur (FeS) clusters in both subunits. Overall, the reactivity of HoxEFU observed here suggests that it functions in managing peripheral electron flow from photosynthetic electron transfer, findings that reveal detailed insights into how ubiquitous cellular components may be used to allocate energy flow into specific bioenergetic products.

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