Characterization of unusual truncated hemoglobins of Chlamydomonas reinhardtii suggests specialized functions.

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CONCLUSION: Annotated hemoglobin genes in Chlamydomonas reinhardtii form functional globins, despite unusual architectures. Spectral characteristics show subtle biochemical differences. Multiple globins might help the alga to cope with its versatile environment. The unicellular green alga C. reinhardtii is a photosynthetic, often soil-dwelling organism, subjected to a changeable environment in nature. The alga contains 12 genes encoding so-called truncated hemoglobins that feature a two-on-two helical fold instead of the three-on-three helix arrangement of the long-studied vertebrate globins or plant symbiotic and non-symbiotic hemoglobins. In plants, non-symbiotic hemoglobins often play a role in acclimation to stress, and we could show recently that one of the C. reinhardtii globin genes is vital for anoxic growth. Here, three further globin encoding transcripts (Cre16.g661000.t1.1, Cre16.g661300.t2.1 and Cre16.g662750.t1.2) were heterologously expressed along with the recently studied THB1. UV-Vis and X-ray absorption spectroscopy analyses show that the sequences indeed encode functional hemoglobins, despite their uncommon primary sequences, which include long C-termini without any predictable function, or a split heme-binding domain. The proteins show some variations regarding the coordination of the heme iron or the interaction with diatomic ligands, indicating different functionalities. The respective transcripts are not responsive to the nitrogen source, in contrast to results reported for THB1, but they accumulate in darkness. This work advances experimental data on the very large globin family in general, and, more specifically, on hemoglobins in photosynthetic organisms.