Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography
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Evaluation Summary:
The manuscript will be of interest to a broad audience in structural biology, biochemistry, and enzymology. The work demonstrates the use of a cutting-edge approach in protein crystallography to investigate and visualize the complex mechanism of an enzyme, the paradigm being Mn-dependent ribonucleotide reductase R2b in complex with flavin-bound NrdI at different redox states. The work is timely and has implications for future investigation of complex biochemical processes.
(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
Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O 2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
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Evaluation Summary:
The manuscript will be of interest to a broad audience in structural biology, biochemistry, and enzymology. The work demonstrates the use of a cutting-edge approach in protein crystallography to investigate and visualize the complex mechanism of an enzyme, the paradigm being Mn-dependent ribonucleotide reductase R2b in complex with flavin-bound NrdI at different redox states. The work is timely and has implications for future investigation of complex biochemical processes.
(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):
The manuscript submitted by Juliane John et al. reported two crystal structures of the Mn-dependent ribonucleotide reductase R2b in complex with flavin-bound NrdI at different redox states. An advanced photoreduction-free crystallographic technique (SFX) was used for crystal diffraction to preserve the structural information reflecting the actual oxidation states of the flavin cofactor. By comparing the hydroquinone- and oxidized quinone-bound complex structures, previous synchrotron structures, and theoretical conformations, the authors showed unbending flavin conformations despite its oxidation state, a binding site relocation near the superoxide-shuttling channel, and protein structural rearrangement around the interaction surface. The results led them to propose that the complex formation restricts …
Reviewer #1 (Public Review):
The manuscript submitted by Juliane John et al. reported two crystal structures of the Mn-dependent ribonucleotide reductase R2b in complex with flavin-bound NrdI at different redox states. An advanced photoreduction-free crystallographic technique (SFX) was used for crystal diffraction to preserve the structural information reflecting the actual oxidation states of the flavin cofactor. By comparing the hydroquinone- and oxidized quinone-bound complex structures, previous synchrotron structures, and theoretical conformations, the authors showed unbending flavin conformations despite its oxidation state, a binding site relocation near the superoxide-shuttling channel, and protein structural rearrangement around the interaction surface. The results led them to propose that the complex formation restricts flavin movement, which changes its redox potential to promote the generation and migration of the superoxide from the flavin oxidation site in NrdI to the di-Mn radical generation site in R2b. The authors claim that the redox potential of flavin induces the structural reorganization of the complex. However, a major concern for this manuscript is that the obtained structures do not represent a consecutive biological process. More investigations are needed to attribute the structural reorganization to the change in the redox states of flavin.
Strengths:
1. The technique of SFX coupled with XEFL provides advantages in determining the crystal structures of the redox-active enzymatic systems, which is a perfect tool to study the target system. All previous structures on this system were obtained with the classic synchrotron X-ray sources on single crystals. The results presented here provide the first views of photoreduction-free structures reflecting the true oxidation states of the flavin cofactor, giving valuable insights into how the flavin cofactor could induce the structural changes and thus, deliver the reactive oxidative species to activate a distant di-Mn center. Such a detailed investigation would be impossible with conventional crystallographic methods.
2. It is a well-done structural study. The structural refinement was nicely conducted and finalized with thorough consideration. For example, the unknown density in close vicinity to the flavin-oxygen interaction site was carefully assessed with different possibilities and analyzed in the context of coordination and surrounding residues, leading to the hypothesis of how superoxide binds and is shuttled rewards R2b. In addition, the use of the isomorphous difference map wisely indicated the differences between the structures of two oxidation states, reducing the model-based bias and pinpointing the most significant structural changes.Weaknesses:
1. Two structures obtained in this study correspond to R2b-NrdI bound with fully reduced and fully oxidized quinone molecules that differ by 2 electrons. The authors compared these structures and attributed the observed structural changes to different redox states of FMN. However, FMN reduction is a one-electron transfer process to generate a single superoxide molecule. Either hydroquinone (hq) is oxidized to semiquinone (sq), or sq is oxidized to the oxidized form (ox), which should be independent of each other. Therefore, without knowing an sq-bound structure, the structural comparison between the hq and ox states does not represent the natural reduction process. In other words, the results presented in this manuscript do not support the proposed mechanism in scheme 3 showing the reduction from hq to sq. The interpretations regarding oxygen reactivity and superoxide positioning could also be overclaiming.
2. A major conclusion is that the structural reorganization is induced by changes in FMN redox state, which is hard to understand considering the FMN conformation shows marginal differences between the oxidized and reduced states. A rationale demonstrating the connection between the redox state and local structural movement is lacking. The previously reported R2b-NrdI complex structures (Boal et al., Science, 2010) obtained using a synchrotron X-ray source also showed structural reorganization at the complex interface between the oxidized and reduced forms (a noticeable loop movement at the position corresponding to the 40s loop). If the reorganization is indeed redox-controlled, such structural differences should not occur in the synchrotron structures since they had already been reduced. Therefore, the structural reorganization may be irrelevant to or not much affected by the redox state of FMN. -
Reviewer #2 (Public Review):
This manuscript focuses on elucidating conformational changes in the structure of the manganese-binding R2b subunit of a Class 1b ribonucleotide reductase in complex with the NrdI flavin-binding protein induced by different oxidation states of the flavin cofactor. Using XFEL to avoid the effects of photoreduction that hindered these goals in previous work, the authors determine structures of the complex in two oxidation states. Analysis of these structures and others reveals that the complex locks the FMN cofactor into a more linear conformation. The authors conclude that the conformational strain induced on the cofactor within the complex drives generation of superoxide radicals by lowering its reduction potential.
Strengths
The major strengths of this study are the use of XFEL to determine structures of …Reviewer #2 (Public Review):
This manuscript focuses on elucidating conformational changes in the structure of the manganese-binding R2b subunit of a Class 1b ribonucleotide reductase in complex with the NrdI flavin-binding protein induced by different oxidation states of the flavin cofactor. Using XFEL to avoid the effects of photoreduction that hindered these goals in previous work, the authors determine structures of the complex in two oxidation states. Analysis of these structures and others reveals that the complex locks the FMN cofactor into a more linear conformation. The authors conclude that the conformational strain induced on the cofactor within the complex drives generation of superoxide radicals by lowering its reduction potential.
Strengths
The major strengths of this study are the use of XFEL to determine structures of the R2b-NrdI complex in multiple oxidation states, which reveals oxidation-induced conformational changes to the FMN cofactor. The authors provide a compelling rationale for their use of the specific methods and analysis they chose that makes the paper understandable to a wide audience.
The structural studies will not only be of impact to people in the field of flavoproteins and ribonucleotide reductases, but also include creative approaches to challenging problems in structural biology.
Beyond the high quality of the structural data and its analysis, the paper is very well-written and also includes a nice discussion of the structures of the manganese dependent enzyme that is the focus of their work to the closely related iron-dependent enzymes.Weaknesses
The hypotheses regarding the mechanism of superoxide generation come entirely from examination of crystal structures, rather than direct experimental measurements -
Reviewer #3 (Public Review):
In this study, the authors were exploring the structural biology and mechanism of Bacillus cereus ribonucleotide reductase R2b-NrdI complex, an essential component of the system for formation of deoxyribonucleotides for DNA synthesis. The R2 subunit relies on two divalent cations (including iron or manganese) for its activity and associates with the Nrdl subunit. Nrdl is a flavoprotein that utilizes the redox activity of FMN to reduce molecular oxygen (O2) to a superoxide radical (O2•-). The superoxide then transits through a channel formed at the interface between Nrdl and R2b.
Previous structure-function studies of this system of enzymes (of which there are many) utilized traditional x-ray diffraction methodologies (i.e. synchrotron radiation) which are damaging to the FMN cofactor and/or divalent cations …
Reviewer #3 (Public Review):
In this study, the authors were exploring the structural biology and mechanism of Bacillus cereus ribonucleotide reductase R2b-NrdI complex, an essential component of the system for formation of deoxyribonucleotides for DNA synthesis. The R2 subunit relies on two divalent cations (including iron or manganese) for its activity and associates with the Nrdl subunit. Nrdl is a flavoprotein that utilizes the redox activity of FMN to reduce molecular oxygen (O2) to a superoxide radical (O2•-). The superoxide then transits through a channel formed at the interface between Nrdl and R2b.
Previous structure-function studies of this system of enzymes (of which there are many) utilized traditional x-ray diffraction methodologies (i.e. synchrotron radiation) which are damaging to the FMN cofactor and/or divalent cations by reducing their oxidation states, preventing a structural picture of the oxidized state of the enzyme complex.
Therefore, the authors utilized serial femtosecond crystallography with an x-ray free electron laser, which allows for room temperature x-ray diffraction data collection. The authors were then able to visualize any structural changes to the flavin ring of FMN between the redox states, any changes in the interaction surface between Nrdl and R2b and the channel between them, and the di-divalent ion binding site of R2b (in this case, manganese). In these regards, the authors were successful in the experiment and solved the FMN oxidized and/or reduced (hydroquinone, FMNH2) states of the Nrdl-R2b complex and could make numerous important observations. Most notably, they observed that the flavin ring of FMN does not undergo major bending between its oxidized and reduced states, which contrasted with previous observations of cryo-cooled synchrotron radiation-solved structure of Nrdl, and with quantum mechanics/molecular mechanics calculations. The evidence presented in terms of quality of the electron density of the FMN moieties in each structure were convincing to me. They also observed that the R2b subunit contributes amino acids that hold the FMN ring in the planar state, suggesting that the Nrdl-R2b complex places strain on the FMN ring allowing favouring of reduction of molecular oxygen to superoxide. The evidence for this observation was also convincing to me and in honesty, it was quite amazing to see the conformational differences in amino acids between the redox states, as nicely presented in electron density difference maps.
Overall, this represents a strong study and excellent example of the utility of serial femtosecond crystallography with an XFEL for providing important mechanistic details into redox-active enzymes. I believe this paper will provide inspiration to the structural biology community to use this approach and provide important appropriate technical details.
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