Selenocyanate derived Se-incorporation into the nitrogenase Fe protein cluster
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Evaluation Summary:
This manuscript describes the unexpected observation of selenium exchange into an iron-sulfur cluster cofactor of a component of nitrogenase. The work sets the stage for future mechanistic study of this phenomenon. It also provides a roadmap for the study of sulfide exchange in other classes of iron-sulfur cluster enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
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 (CO 2 ) reduction and facilitating MoFe protein metallocluster biosynthesis. The precise role(s) of the Fe protein Fe 4 S 4 cluster in some of these processes remains ill-defined. Herein, we report crystallographic data demonstrating ATP-dependent chalcogenide exchange at the Fe 4 S 4 cluster of the nitrogenase Fe protein when potassium selenocyanate is used as the selenium source, an unexpected result as the Fe protein cluster is not traditionally perceived as a site of substrate binding within nitrogenase. The observed chalcogenide exchange illustrates that this Fe 4 S 4 cluster is capable of core substitution reactions under certain conditions, adding to the Fe protein’s repertoire of unique properties.
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Evaluation Summary:
This manuscript describes the unexpected observation of selenium exchange into an iron-sulfur cluster cofactor of a component of nitrogenase. The work sets the stage for future mechanistic study of this phenomenon. It also provides a roadmap for the study of sulfide exchange in other classes of iron-sulfur cluster enzymes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this effort, the authors investigate whether sulfide ions in the nitrogenase Fe protein Fe4S4 cluster can be exchanged with selenide ions using x-ray crystallographic detection of the newly incorporated Se. Strengths include the quality of the datasets collected and the thorough nature of the experimental design. The results are convincing and well-validated. Weaknesses include a narrow scope with broader applications that may not be sufficiently defined for a general audience.
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Reviewer #2 (Public Review):
In this study, Buscagan et al. describe the ATP-dependent replacement of the sulfur components of the nitrogenase iron protein 4Fe-4S cluster by selenium, as determined by X-ray crystallographic analyses. The iron protein is quite distinct from canonical 4Fe-4S ferredoxins in that a) its reactivity and redox activity is modulated by ATP, b) it can alternate between three oxidation states, and c) it serves multiple functional roles, ranging from electron transfer to support nitrogen fixation to cofactor maturation. It has also been shown to be involved in CO2 reduction. Thus, the chemistry of the iron protein 4Fe-4S cluster and its systematic modification are of substantial interest. The authors have previously shown that the catalytic cluster of nitrogenase can readily undergo S-to-Se exchange reactions …
Reviewer #2 (Public Review):
In this study, Buscagan et al. describe the ATP-dependent replacement of the sulfur components of the nitrogenase iron protein 4Fe-4S cluster by selenium, as determined by X-ray crystallographic analyses. The iron protein is quite distinct from canonical 4Fe-4S ferredoxins in that a) its reactivity and redox activity is modulated by ATP, b) it can alternate between three oxidation states, and c) it serves multiple functional roles, ranging from electron transfer to support nitrogen fixation to cofactor maturation. It has also been shown to be involved in CO2 reduction. Thus, the chemistry of the iron protein 4Fe-4S cluster and its systematic modification are of substantial interest. The authors have previously shown that the catalytic cluster of nitrogenase can readily undergo S-to-Se exchange reactions under catalytic turnover conditions using potassium selenocyanate (KSeCN) as a Se source. Here the authors show through anomalous X-ray diffraction analyses that KSeCN can also enable the partial substitution of the S atoms of the iron protein 4Fe-4S cluster by Se. Interestingly, the S-Se substitution is only possible in the presence of ATP which is well known to change both the reduction potential and the chemical reactivity of the iron protein 4Fe-4S cluster. The authors also show that the EPR spectrum of the Se-substituted cluster is similar to that of the native cluster in the presence of dithionite (with g=2 signal indicative of S=1/2 signal of [4Fe-4S/Se]+1 oxidation state). In general, the conclusions of the paper are well supported by the experiments. This study demonstrates that, like the iron-molybdenum cofactor of nitrogenase, the iron protein 4Fe-4S cluster is dynamic in nature and capable of undergoing substantial structural transformations. As such, I don't have any suggested changes to the X-ray or EPR analysis.
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