The Structure and Reactivity of the HoxEFU Complex from the Cyanobacterium Synechocystis sp. PCC 6803

Jacob Artz, Monika Tokmina-Lukaszewska, David Mulder, Carolyn Lubner, Kirstin Gutekunst, Jens Appel, Brian Bothner, Marko Boehm, Paul King

Research output: Contribution to journalArticlepeer-review

12 Scopus Citations

Abstract

Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H2)-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 H2, 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 functioning in vitro as a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD1 and NADP1 were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)1, Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD1. 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.

Original languageAmerican English
Pages (from-to)9445-9454
Number of pages10
JournalJournal of Biological Chemistry
Volume295
Issue number28
DOIs
StatePublished - 2020

Bibliographical note

Publisher Copyright:
© 2020 Artz et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.

NREL Publication Number

  • NREL/JA-2700-76164

Keywords

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

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