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

Abstract

<jats:p>Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H<jats:sub>2</jats:sub>)-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<jats:sub>2</jats:sub>, 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 <jats:italic>in vitro</jats:italic> as a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD<jats:sup>+</jats:sup> and NADP<jats:sup>+</jats:sup> were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)<jats:sup>+</jats:sup>, Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD<jats:sup>+</jats:sup>. 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.</jats:p>

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO 2 step‐down

“…From possible electron sinks only HoxU, the diaphorase subunit of the bidirectional hydrogenase was enhanced in Δ flv1 /Δ flv3 cells (Table 3). However, we did not detect the other subunits of the hydrogenase (Artz et al, 2020).…”

Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria

“…Among others, as shown in this study, high NADH/NAD + ratios can inactivate enzymes that rely on this couple and thereby support the action of isoenzymes that interact with the Fx red /Fx ox couple instead. In addition, electron turntables as the transhydrogenase, FNR and the diaphorase can support this shift (26, 46).…”

“…Electrons from NADPH could be further transferred to ferredoxin via ferredoxin-NADPH-oxidoreductase (FNR). Another potential turntable for the exchange of electrons is the diaphorase part of the NiFe-hydrogenase in Synechocystis, which was recently shown to shuttle electrons between NAD(P)H, flavodoxin and several ferredoxins in vitro (26). Electrons from glucose oxidation that arrive at PSI require ferredoxin-dependent photosynthetic complex I (NDH-1).…”

“…PCC 7942 and Synechocystis was therefore surprising but is well in line with the observation that other enzymes assigned to anaerobic metabolism in eukaryotes are expressed in the presence of oxygen as well (10, 24). Synechoystis possesses a network of up to 11 ferredoxins containing 2Fe2S, 3Fe4S and 4Fe4S clusters (25, 26). The 2Fe2S ferredoxin 1 (Ssl0020) is essential and by far the most abundant ferredoxin in Synechocystis and is the principal acceptor of photosynthetic electrons at PSI (27).…”

“…The 2Fe2S ferredoxin 1 (Ssl0020) is essential and by far the most abundant ferredoxin in Synechocystis and is the principal acceptor of photosynthetic electrons at PSI (27). Structures, redox potentials and distinct functions have been resolved for some of the alternative low abundant ferredoxins, however, the metabolic significance of the complete network is still far from being understood (25,26,(28)(29)(30)(31).…”