Resistance of Nitric Oxide Dioxygenase and Cytochrome c Oxidase to Inhibition by Nitric Oxide and Other Indications of the Spintronic Control of Electron Transfer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an excellent review that embodies a unique and informed perspective on the mechanisms and pathways for NO inhibition of two important enzyme classes.

The author has contributed significant discoveries to the field over the years, and this review provides an informed historical context to the new discoveries that have emerged over the last several years.

Of particular significance to this review is the incorporation of the recently hypothesized “ferric + dioxygen” interaction (e.g., section 3.3.4). In the normal chemical literature, such an interaction has been discounted on the grounds that the ferric ion does not have sufficient electron density to back-donate into the pi antibonding orbitals to create a strong Fe-O interaction (similar to that seen in the ferrous state). The author has done a nice job in incorporating the electrostatic features of this interaction into his perspective on the relevance of such a species. In this reviewer’s opinion, this has been very nicely done and provides a unique perspective that will ignite insightful discussions in the field. To this end, this review is timely and is much needed!

The referencing is very detailed and informative. The depth of referencing is quite impressive, actually.  For example, incorporating the often-forgotten work of Brudvig and Chan (ref 86) and other earlier researchers really makes this review a treasure trove to the vast field of researchers in this area of work.

Finally, the author appears to have been very careful to emphasize that this review contains some perspectives, and this approach makes it very interesting and valuable to read.

Minor suggestions for improvement: 

       The Abstract reads more like a “summary and justification for the work” rather than an Abstract that highlights the important contributions contained in this review.  This may be this reviewer’s preference, but the authors may want to consider improving the content for the Abstract.

Author Response

Please see attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a comprehensive review paper that proposes novel mechanisms for NO resistance in two important oxygen-metabolizing enzyme systems: nitric oxide dioxygenases and cytochrome c oxidase (NOD and CcO, respectively). While the paper addresses an important biological paradox and presents creative theoretical solutions, it has significant limitations that affect its suitability for publication in the present state. The paper tackles a real and important contradiction: how can enzymes that should be irreversibly inhibited by NO (based on measured binding affinities) actually show reversible, competitive inhibition? This is a legitimate scientific puzzle worthy of investigation. The Author demonstrates extensive knowledge of the field, citing over 230 references and providing thorough coverage of relevant research spanning decades.The proposed "spintronic" mechanism involving spin-orbital coupling and electron-transfer switching represents a creative theoretical thinking that could stimulate new research directions. Although the paper provides detailed molecular mechanisms with specific structural predictions, it is not clear if the hypotheses are testable.

Major Concerns
A) Lack of direct experimental evidence. The paper's fundamental weakness is that it presents entirely theoretical models without providing new experimental data to support the proposed mechanisms. The "spintronic" electron-transfer switching mechanism, while creative, lacks direct experimental validation. The use of "spintronics" borrowed from materials science may be confusing to biochemical audiences without adequate explanation.
B) Speculative nature of key claims. Critical assertions, such as the existence of ferric heme-O2 complexes in the proposed catalytic cycles, are largely theoretical. As far as this reviewer knows, there is overwhelming experimental and theoretical evidence against this hypothesis. The paper relies heavily on indirect evidence and analogies rather than direct biochemical proof.
C) Overreaching conclusions. The Author claims to have "resolved" longstanding paradoxes and that "progress on many fronts has illuminated" the mechanisms. However, the evidence presented is insufficient to support such definitive statements.
D) Complex theoretical models without validation. In the cases of NOD and CcO the proposed catalytic cycles (Figures 1 and 5-8) are highly detailed but represent untested hypotheses. The paper would benefit from computational modeling or experimental predictions that could validate these mechanisms.
E) Writing and presentation issues. The paper is extremely long (44 pages) and could be more concise. Some sections are overly speculative. The transition between established facts and novel hypotheses could be clearer
F) Thermodynamic and kinetic considerations. While the paper discusses energetics, more rigorous thermodynamic analysis of the proposed pathways would strengthen the arguments. The kinetic arguments, while extensive, sometimes rely on estimates and approximations that may not be sufficiently rigorous. Although structural information is discussed, the paper could benefit from more detailed analysis of how the proposed mechanisms align with high-resolution structural data.

Minor Issues
1. Figure quality: some figures (particularly the catalytic cycle diagrams) are complex and could be simplified for better comprehension.
2. Citation format: the reference formatting is inconsistent in places.

Author Response

Please see attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The review has clearly improved; nonetheless, the Author has not addressed the following point. In my first major concern, point B) I pointed out the lack of evidence for the binding of O2 to BNCox. The author cites ref 186 to partially support his hypothesis; however, he overlooks the following: 1) the experiments in ref 186 actually provide direct contradictory evidence against O2 binding to the oxidized BNC (Fig 6 of ref 186). The optical spectroscopy measurements directly tested for O2 binding by comparing the UV-VIS spectra of oxidized CcO under anaerobic versus aerobic conditions. The fact that "no spectral difference was observed" is direct experimental evidence that argues against significant O2 binding to the oxidized BNC under the tested conditions. 2) the DFT calculations, while sophisticated, rely on theoretical models with inherent uncertainties. The Authors in ref. 186 acknowledge that DFT "is not considered sufficiently accurate to discriminate the energetical ordering" of different spin states, and that binding energies depend significantly on the choice of density functional. 3) A seminal study by Antonini et al. (1970, Nature, 228, 936–937) demonstrated that reoxidation of partially reduced CcO proceeds slowly, suggesting that O2 binding to the BNC occurs only after at least two electrons have been transferred to BNCox.

Furthermore, from a purely mechanistic perspective, the proposed CcO catalytic cycle (Fig. 5) implausibly suggests that electron transfer to and within CcO cannot occur unless O₂ is already bound.

The Author should take the above considerations into account.

Author Response

The review has clearly improved; nonetheless, the Author has not addressed the following point. In my first major concern, point B) I pointed out the lack of evidence for the binding of O2 to BNCox. The author cites ref 186 to partially support his hypothesis; however, he overlooks the following: 1) the experiments in ref 186 actually provide direct contradictory evidence against O2 binding to the oxidized BNC (Fig 6 of ref 186). The optical spectroscopy measurements directly tested for O2 binding by comparing the UV-VIS spectra of oxidized CcO under anaerobic versus aerobic conditions. The fact that "no spectral difference was observed" is direct experimental evidence that argues against significant O2 binding to the oxidized BNC under the tested conditions.

RESPONSE:  I have added and made changes to the following paragraph to make the important results and possible interpretations clearer to the reader:  “The failure of Kaila et al. [186] to observe spectral differences in CcO BNC_ox with 280 µM O₂ may be explained by the uptake of excess protons by the putative Pro tunnel (Section 4.3.4, Fig. 8) and a hydrated BNC_ox (O_W).  In the absence of an opposing membrane potential, protonation of the a₃ propionates would decrease the electron density of the heme required for O₂ binding.  Indeed, the CcO Soret maxima (Kaila et al., Fig. 6) appears blue-shifted at ~421 nm vs. the ~424 nm recorded by Chance et al. [251] which may occur with propionate carboxylate protonation, hydration, or an increase in high-spin iron [74].  Water bound to the BNC_ox (O_W) may also cause a failure to observe the proposed O₂ SOC.  Dehydrating BNC_ox to an O intermediate state [163] may be essential for significant O₂ interaction.  A similar water and O₂ competition is apparent in ferric Mb [74].”

I also made changes to an earlier statement concerning the results (DFT and spectra) and interpretations of Kaila et al.:

“Apparently, the authors did not grasp the implication of univalent O₂ reduction (Fig. 1A in Kaila et al.) for CcO catalysis (see Fig. 5) or rejected the possibility due to i) thermodynamic considerations, ii) a failure to observe UV-visible spectral changes (Fig. 6 in Kaila et al.), iii) an unawareness of the role of SOC in redox reactions and the burgeoning field of spintronics, and iv) unforeseen mechanisms for the membrane potential to affect proton tunneling, O_W (or O_H) dehydration, and ET (see below).”

I have also added the following statement in the initial description of the structural model in Figure 8:

“The Pro tunnel may also serve in water removal and ‘dry out the surroundings of the BNC_ox‘ in O_W (aka O_H) (Fig. 5) to form the dehydrated O intermediate [163] which would be expected to be more accessible to O₂.  An electrostatically-driven water pumping or efflux mechanism that depends on the series of water dipole rotations with proton hopping [223], the alpha-helix dipole, the local membrane potential, and any cycle-dependent opening and closing motions of the Asp486-Thr488 stricture can now be modelled.  The Pro tunnel, or water pump, may also replenish the water required to sustain fatty acid oxidation and other matrix-localized water-consuming metabolic pathways.”

In the paragraph describing possible experiments, I add:  “Does C¹⁸O₂ formation via ¹⁸O₂ respiration, H₂¹⁸O formation, and fatty acid oxidation depend on CcO and the putative Pro tunnel or (de)hydrating water pump?”

2) the DFT calculations, while sophisticated, rely on theoretical models with inherent uncertainties. The Authors in ref. 186 acknowledge that DFT "is not considered sufficiently accurate to discriminate the energetical ordering" of different spin states, and that binding energies depend significantly on the choice of density functional.

RESPONSE:  I have added “albeit tentative” to indicate that the conclusions of the studies are indefinite in the statement: “Furthermore, DFT calculations, albeit tentative, support weak O₂ binding to BNC_ox [186].”

3) A seminal study by Antonini et al. (1970, Nature, 228, 936–937) demonstrated that reoxidation of partially reduced CcO proceeds slowly, suggesting that O2 binding to the BNC occurs only after at least two electrons have been transferred to BNCox.

Furthermore, from a purely mechanistic perspective, the proposed CcO catalytic cycle (Fig. 5) implausibly suggests that electron transfer to and within CcO cannot occur unless O₂ is already bound.

The Author should take the above considerations into account.

RESPONSE:  I have considered the results of Antonini et al. and the referee’s and readers’ potential viewpoint with the following changes:  “In addition, the proposed ET switch would also prevent the untimely reduction of the ferryl intermediate (F_H), formation of highly reactive hydroxyl radical, and potentially irreversible damage to CcO.  Where a slow phase has been observed, and the electron supply does not appear limiting and may be dysregulated [234] or the O₂ SOC may be impaired by a hydrated (O_W) and protonated BNC_ox, the reversibility of function should be examined and the damage, protonation, and hydration assessed.  Given the observed single turnover fast to slow phase transition [234], a failed dehydrating Ow to O cycle step that occurs with a non-existent membrane potential in the isolated CcO with possible Pro tunnel/pump dysfunction appears most attractive.  Not recognizing, or accounting for, the existence of a sensitive and rapid ET gate or switch (Fig. 7) and/or a proton uptake and water expulsion tunnel (Fig. 8) and O_W (O_H) dehydration mechanism would cause incalculable confusion.  For well over 50 years, the fast-slow phase a₃ reduction phenomenon [234] has been interpreted as evidence for the requirement of a 2 e- reduction of the BNC for O₂ binding [159, 163].  The NO sensitivity-resistance of CcO discussed in Sections 4.2 and 4.3 and the paucity of R in respiring mitochondria [101, 102] strongly suggests otherwise. ”

 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

My compliments to the Author.