Kaiser, J. T. | bioRxiv Science

bioRxiv2022

Selenocyanate Derived Se-Incorporation into the Nitrogenase Fe Protein Cluster

The nitrogenase Fe protein mediates ATP-dependent electron transfer to the nitrogenase MoFe protein during nitrogen fixation, in addition to catalyzing MoFe protein independent substrate (CO2) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe4S4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe4S4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source. The observed chalcogenide exchange illustrates that this Fe4S4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe proteins repertoire of unique properties.

biochemistry↗

bioRxiv2019

Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter

ATP Binding Cassette (ABC) transporters often exhibit significant basal ATPase activity in the absence of transported substrates. To investigate the factors that contribute to this inefficient coupling of ATP hydrolysis to transport, we characterized the structures and functions of variants of the bacterial Atm1 homolog from Novosphingobium aromaticivorans (NaAtm1), including forms with disulfide crosslinks between the nucleotide binding domains. Unexpectedly, disulfide crosslinked variants of NaAtm1 reconstituted into proteoliposomes not only transported oxidized glutathione, but also exhibited more efficient coupling of ATP hydrolysis to GSSG transport than the native transporter. These observations suggest that enhanced conformational dynamics of reconstituted NaAtm1 may contribute to the inefficient use of ATP. Understanding the origins of this uncoupled ATPase activity, and reducing the impact through disulfide crosslinking or other protocols, will be critical for the detailed dissection of ABC transporter mechanism to assure that the ATP dependent steps are indeed relevant to substrate translocation.

biochemistry↗