Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron–sulfur‐containing, electron‐transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin‐like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His‐tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron–sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be −386 ± 4 mV, −386 ± 2 mV, −559 ± 10 mV and −557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2. Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui. Ferredoxins are key proteins in anaerobic metabolism. In early life forms such as the acetogenic bacteria, low potential ferredoxins serve to reduce carbon dioxide. Acetogens contain multiple genes each potentially encoding a different ferredoxin, but this and their function in metabolism remained elusive. Here, we have identified, purified and characterized four ferredoxins from the thermophilic, acetogenic model organism Thermoanaerobacter kivui and found two to be involved in CO2 reduction.