Expression of two [Fe]‐hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions

Abstract

We have isolated and characterized a second [Fe]-hydrogenase gene from the green alga, Chlamydomonas reinhardtii. The HydA2 gene encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [Fe]-hydrogenases. We demonstrate that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Furthermore, the expression levels of both transcripts are regulated (in some cases differently) by incubation conditions, such as the length of anaerobiosis, the readdition of O 2 , the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H 2 -gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe-S centers and the existence of extra sequences in the algal enzymes. To our knowledge, this work represents the first systematic study of expression of two algal [Fe]hydrogenases in the same organism.

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…In this range of light intensities, growth and photosynthesis of aerated cultures were similar for all the strains (Table I). After 1 h of acclimation to dark and anoxia, a time required for proper expression and activity of hydrogenases (Forestier et al, 2003;Godaux et al, 2013;Supplemental Fig. S2), we measured the hydrogen evolution rate (J H2 ) and the quantum yield of PSII (f PSII ) and PSI (f PSI ), as well as the mean photochemical rate (R ph ) based on electrochromic shift (ECS) measurements at the onset of light (10 s; Table II).…”

“…The only exception to this rule is the low ETR in pgrl1 after 10 min of anoxia (Fig. 5A), which is probably due to the fact that the hydrogenases are not yet fully expressed (Forestier et al, 2003;Pape et al, 2012), and therefore mimics the behavior of pgrl1 hydg-2.…”

Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase

“…These sites are connected to the surface by a hydrophobic channel that facilitates H 2 diffusion. The additional [FeS] clusters required for electron transport from/to soluble mediators are also found in most [Fe] hydrogenase enzymes (2,15,16); however, algal [Fe] hydrogenases lack these additional metal clusters (19,20).…”

Cloning and structure analysis of hydrogenase gene from Chlamydomonas reinhardtii SE

“…The primers for RT-PCR were synthesized and based on the published sequence of C. reinhardtii 21gr [7]. The total RNA of C. reinhardtii SE was used as a template for RT-PCR amplification, performed with primers of F 1 /R 1 .…”

“…The gene structure and expression of the hydrogenase has been studied. So far, hydrogenase genes of many algaes have been reported, including hydA of the Scenedesmus obliquus, hydA of the Chllorella fiscal, hydA1 of C. reinhardtii, and hydA2 of CC-425 [4][5][6][7]. There were many reports about hydA2.…”