Protonation/deprotonation-driven switch for the redox stability of low-potential [4Fe-4S] ferredoxin

Reviewer #1 (Public review):

Summary:

The authors introduced neutron crystallography coupled with room temperature X-ray crystallography to exam the redox properties of the BtFt [4Fe-4S] cluster expressed in E. coli. Neutron structure allowed the authors to exam the influence of Asp64 on the redox properties of the [4Fe-4S] cluster. The neutron structure also allowed for the identification of the hydrogen network around the [4Fe-4S] structure. This work was followed by density functional theory calculation to examine different redox states which also pointed to the role of Asp64 in affecting or dictating redox function of the [4Fe-4S] cluster. Based on the DFT work the authors examine the redox properties under oxic and anoxic conditions in wild type enzymes and in a D64N mutant again showing the role of Asp64 on the redox kinetics and redox potential of the [4Fe-4S] cluster. Lastly, the authors examined similar [4Fe-4S] ferredoxins from several organisms and with a Asp64 or Glu64 observed a similar role of Asp64 on the low potential state of the [4Fe-4S] cluster. The major conclusion of the study was to identify the role of specific amino acids, in this case Asp64, in controlling the redox state and kinetics of [4Fe-4S] clusters. The authors also demonstrate the strength of neutron crystallography when combined with classical X-ray crystallography and classical spectral/redox studies.

Strengths:

In general, the experimental design is logical and the results are convincing demonstrating the role of Asp64 on the redox properties of [4Fe-4S] clusters in ferredoxins.

Weaknesses:

The role(s) of coordinating amino acids on the redox properties of a functional group is not surprising, this reviewer believes this is a novel result in ferredoxins and does make a nice contribution to the field.

Reviewer #2 (Public review):

In this study, Wada et al. investigate the low potential ferredoxin from Bacillus thermoproteolyticus (BtFd) using a combination of neutron crystallography, x-ray crystallography, DFT and spectroscopy to determine the influence of hydrogen bonding networks on the redox potential of ferredoxin's 4Fe-4S cluster. The use of neutron diffraction allowed the authors to probe the precise location of hydrogens around the 4Fe-4S cluster, which was not possible from prior studies, even with the previously reported high-resolution (0.92 Å) structure of BtFd. This allowed the authors to revise prior models of the proposed H bonding network theorized from earlier x-ray crystallography studies ( for example, showing that there is not in fact a H bond formed between the Thr63-O𝛾1 and the [4Fe-4S]-S4 atoms). With this newly described H-bonding network established, the electronic structure of the 4Fe-4S cluster was then investigated using DFT methodology, revealing a startling role of the deprotonated surface residue Asp64, which bears substantial electronic density in the LUMO which is otherwise localized to the 4Fe-4S cluster. While aspartate is usually deprotonated at physiological pH, the authors provide compelling evidence that this aspartate has a much higher pKa than is usual, and is able to act as a protonation-dependent switch which controls the stability of the reduced state of the 4Fe-4S cluster, and thus the redox potential.

The findings of this study and the conclusions drawn from them are well supported by the data and computational work. Their findings have implications for similar control mechanisms in other, non-ferredoxin 4Fe-4S bearing electron transport proteins which have yet to be explored, providing great value to the metalloprotein community. One change that the authors may consider to enhance the clarity of the manuscript regards the nomenclature used for the varying models discussed (CM, CMNA, CMH and so forth). It would be beneficial to the reader if the nomenclature included the redox state (ox. vs red.) of the model in the model's name.