A Nonmitochondrial Hydrogen Production in Naegleria gruberi

Abstract

Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes.

Diversity and origins of anaerobic metabolism in mitochondria and related organelles

“…HYD, HYD maturases and ASCT1B and 1C were identified in the genome, and were predicted to function in mitochondria ( possibly in hypoxic conditions) based on the presence of predicted N-terminal targeting sequences [42], although a recent report argues that hydrogen production occurs exclusively in the cytoplasm [43]. [18,[48][49][50].…”

Adaptive iron utilization compensates for the lack of an inducible uptake system inNaegleria fowleriand represents a potential target for therapeutic intervention

“…The most fundamental finding of this experiment is that mainly cytosolic iron-containing proteins were downregulated. These are components of probably nonessential pathways, such as the phenylalanine degradation pathway, hydrogenase maturation and hydrogen production; the latter is surprisingly shown to take place in the cytosol of N. gruberi [32]. On the other hand, mitochondrial iron-dependent proteins were generally unchanged, while some components of the iron-sulfur cluster synthesis machinery were upregulated in iron-deficient conditions, emphasizing the essential role of the iron-dependent respiration process.…”

Biology and pathogenesis of Naegleria fowleri

“…The approximate intervals of G1, S, G2, and M phases are 6% M phase (28 min), 38% G1 phase (180 min), 38% S phase (183 min and succinate oxidation (207). The genome of N. gruberi showed its versatility, and although aerobic, its genome predicts anaerobic respiration (204,208,210). N. gruberi has a functional…”

“…[FeFe]-hydrogenase, as determined by measuring hydrogen production (210). Hydrogenase enzyme is accredited to anaerobic organisms.…”