X-Ray Structure and enzymatic study of a Bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

Reviewer #1 (Public Review):

Summary:
The manuscript describes the crystal structures of Streptococcus pneumoniae NOXs. Crystals were obtained for the wild-type and mutant dehydrogenase domain, as well as for the full-length protein comprising the membrane domain. The manuscript further carefully studies the enzyme's kinetics and substrate-specificity properties. Streptococcus pneumoniae NOX is a non-regulated enzyme, and therefore, its structure should provide a view of the NOX active conformation. The structural and biochemical data are discussed on this ground.

Strengths:
This is very solid work. The protein chemistry and biochemical analysis are well executed and carefully described. Similarly, the crystallography must be appreciated given the difficulty of obtaining good enzyme preparations and the flexibility of the protein. Even if solved at medium resolution, the crystal structure of the full-length protein conveys relevant information. The manuscript nicely shows that the domain rotations are unlikely to be the main mechanistic element of NOX regulation. It rather appears that the NADPH-binding conformation is pivotal to enzyme activation. The paper extensively refers to the previous literature and analyses the structures comprehensively with a comparison to previously reported structures of eukaryotic and prokaryotic NOXs.

Weaknesses:
The manuscript is not always very clear with regard to the analysis of NADPH binding. The last section describes a "crevice" featured by the NADPH-binding sites in NOXs. It remains unclear whether this element corresponds to the different conformations of the protein C-terminal residues or more extensive structural differences. This point must be clarified.
A second less convincing point concerns the nature of the electron acceptor. The manuscript states that this NOX might not physiologically act as a ROS producer. A question then immediately arises: Is this protein an iron reductase? Can the authors better discuss or provide more data about this point?

Reviewer #2 (Public Review):

The authors describe the structure of the S. pneumoniae Nox protein (SpNOX). This is a first. The relevance of it to the structure and function of eukaryotic Noxes is discussed in depth.

Strengths and Weaknesses
One of the strengths of this work is the effort put into preparing a pure and functionally active SpNOX preparation. The protein was expressed in E. coli and the purification and optimization of its thermostability and activity are described in detail, involving salt concentration, glycerol concentration, and pH.

This reviewer was surprised by the fact that the purification protocol in THIS paper differs from those in the mBio and Biophys. J. papers by the absence of the detergent lauryl maltose neopentyl glycol (LMNG). LMNG is only present in the activity assay at a low concentration (0.003%; molar data should be given; by my calculation, this corresponds to 30 μM).

In light of the presence of lipids in cryo-EM-solved structures of DUOX and NOX2, it is surprising that the authors did not use reconstitution of the purified SpNOX in phospholipid (nanodisk?). The issue is made more complicated by the statement on p. 18 of "structures solved in detergent like ours" when no use of detergent in the solubilization and purification of SpNOX is mentioned in the Methods section (p. 21-22).

Can the authors provide information on whether E. coli BL21 is sufficiently equipped for the heme synthesis required for the expression of the TM domain of SpN NOX. Was supplementation with δ-aminolevulinic acid used?

The 3 papers on SpNOX present more than convincing evidence that SpNOX is a legitimate Nox that can serve as a legitimate model for eukaryotic Noxes (cyanide resistance, inhibition by DPI, absolute FAD dependence, and NADPH/NADH as the donor or electrons to FAD). It is also understood that the physiological role of SpNOX in S. pneumoniae is unknown and that the fact that it can reduce molecular oxygen may be an experimental situation that does not occur in vivo.

I am, however, linguistically confused by the statement that "SpNOX requires "supplemental" FAD". Noxes have FAD bound non-covalently and this is the reason that, starting from the key finding of Babior on NOX2 back in 1977 to the present, FAD has to be added to in vitro systems to compensate for the loss of FAD in the course of the purification of the enzyme from natural sources or expression in a bacterial host. I wonder whether this makes FAD more of a co-substrate than a prosthetic group unless what the authors intend to state is that SpNOX is not a genuine flavoprotein.

I am also puzzled by the statement that SpNOX "does not require the addition of Cyt c to sustain superoxide production". Researchers with a Cartesian background should differentiate between cause and effect. Cyt c serves merely as an electron acceptor from superoxide made by SpNOX but superoxide production and NADPH oxidation occur independently of the presence of added Cyt c.

The ability of the DH domain of SpNOX (SpNOXDH) to produce superoxide is surprising to this reviewer. The result is based on the inhibition of Cyt c reduction by added superoxide dismutase (SOD) by 40%. In all eukaryotic Noxes superoxide is produced by the one-electron reduction of molecular oxygen by electrons originating from the distal heme, having passed from reduced FAD via two hemes. The proposal that superoxide is generated by direct transfer of electrons from FAD to oxygen deserves a more in-depth discussion and relies too heavily on the inhibitory effect of SOD. A control experiment with inactivated SOD should have been done (SOD is notoriously heat resistant and inactivation might require autoclaving).

An unasked and unanswered question is that, since under aerobic conditions, both direct Cyt c reduction (60%) and superoxide production (40%) occur, what are the electron paths responsible for the two phenomena occurring simultaneously?

This reviewer had difficulty in following the argument that the fact that the kcat of SpNOX and SpNOXDH are similar supports the thesis that the rate of enzyme activation is dependent on hydride transfer from nicotinamide to FAD.

The section dealing with mutating F397 is a key part of the paper. There is a proper reference to the work of the Karplus group on plant FNRs (Deng et al). However, later work, addressing comparison with NOX2, should be cited (Kean et al., FEBS J., 284, 3302-3319, 2017). Also, work from the Dinauer group on the minimal effect of mutating or deleting the C-terminal F570 in NOX2 on superoxide production should be cited (Zhen et al., J. Biol. Chem. 273, 6575-6581, 1998).

It is not clear why mutating F397 to W (both residues having aromatic side chains) would stabilize FAD binding. Also, what is meant by "locking the two subdomains of the DH domain"? What subdomains are meant?

Methodological details on crystallization (p. 11) should be delegated to the Methodology section. How many readers are aware that SAD means "Single Wavelength Anomalous Diffraction" or know what is the role of sodium bromide?

The data on the structure of SpNOX are supportive of a model of Nox activation that is "dissident" relative to the models offered for DUOX and NOX2 activation. These latter models suggested that the movement of the DH domain versus the TM domain was related to conversion from the resting to the activated state. The findings reported in this paper show that, unexpectedly, the domain orientation in SpNOX (constitutively active!) is much closer to that of resting NOX2. One of the criteria associated with the activated state in Noxes was the reduction of the distance between FAD and the proximal heme. The authors report that, paradoxically, this distance is larger in the constitutively active SpNOX (9.2 Å)
than that in resting state NOX2 (7.6 Å) and the distance in Ca2+-activated DUOX is even larger (10.2 Å).

A point made by the authors is the questioning of the paradigm that activation of Noxes requires DH domain motion. Instead, the authors introduce the term "tensing", within the DH domain, from a "relaxed" to a more rigid conformation. I believe that this proposal requires a somewhat clearer elaboration.

The statement on p. 18, in connection to the phospholipid environment of Noxes, that the structure of SpNOX was "solved in detergent" is puzzling since the method of SpNOX preparation and purification does not mention the use of a detergent. As mentioned before, this absence of detergent in the present report was surprising because LMNG was used in the methods described in the mBio and Biophys. J. papers. The only mention of LMNG in the present paper was as an addition at a concentration of 0.003% in the activity assay buffers.

The Conclusions section contains a proposal for the mechanism of conversion of NOX2 from the resting to the activated state. The inclusion of this discussion is welcome but the structural information on the constitutively active SpNOX can, unfortunately, contribute little to solving this important problem. The work of the Lambeth group, back in 1999 (cited as Nisimoto et al.), on the role of p67-phox in regulating hydride transfer from NADPH to FAD in NOX2 may indeed turn out to have been prophetic. However, only solving the structure of the assembled NOX2 complex will provide the much-awaited answer. The heterodimerization of NOX2 with p22-phox, the regulation of NOX2 by four cytosolic components, and the still present uncertainty about whether p67-phox is indeed the final distal component that converts NOX2 to the activated state make this a formidable task.
The work of the Fieschi group on SpNOX is important and relevant but the absence of external regulation, the absence of p22-phox, and the uncertainty about the target molecule make it a rather questionable model for eukaryotic Noxes. The information on the role of the C-terminal Phe is of special value although its extension to the mechanism of eukaryotic Nox activation proved, so far, to be elusive.