Was H₂O₂ generated before oxygenic photosynthesis?

Abstract

We obviously agree with Wu et al. that H₂O₂ might accumulate in the Archean land waters devoid of Fe²⁺. We do disagree on the topic of the half-life of H₂O₂, as the work cited in support for a longer half-live is not relevant to the conditions in the Archean ocean. While the existence of radicals in quartz is not in doubt, we do question the hypothesis that these radicals oxidize water to HO^• and H₂O₂.

As illustrated by the comment from Wu et al. [1], the question as to when O₂ and H₂O₂ were introduced during the Earth's history is an emerging research topic. We agree with Wu et al. [1] that at locations devoid of Fe²⁺ the efficient reduction of H₂O₂ by Fe²⁺ would, obviously, not occur. The title of our publication [2] emphasizes that our focus is on "the important role of iron(II) in the Archean ocean", and we stated explicitly that "H₂O₂ would only survive in water without iron or other redox-active metals" [2]. Thus, we do not disagree with Wu et al. [1] on this issue.

Wu et al. [1] contrast the estimated half-life of 0.7 s [2] with a value of 3.6 h as deduced from Fig.1a shown in their comment. With due respect, we question the scientific reasoning by Wu et al. here. Fig.1a was not published by Wu et al. [1], it was taken from a paper by Wang [3]. The topic of that paper is unrelated to the geochemical Archean issue: Wang [3] investigated the disappearance of an azo dye and showed that 540 μM H₂O₂ at pH 6.4 is stable in the presence of 5 μM Fe²⁺, has a half-life of 3.6 h with 16 μM Fe²⁺, and vanishes instantly at 34 μM Fe²⁺. Compared to the conditions in the Archean ocean, the differences in concentrations, kinetics, pH, and that HCO₃⁻ catalyzes the Fenton reaction (see Ref. [4] for review), are such that we doubt whether Fig.1a usefully contributes to the discussion. Curiously, a nearly identical graph with Fe³⁺ is shown in that publication of Wang [3], which Wu et al. [1] do not mention. The calibration curve shown in Fig.1b of Wu et al. [1] has nothing to do with the data in Fig.1a. Taken together, it is difficult to see what Fig.1a and b convey.

We would like to express some reservations regarding the method employed by Wu and coworkers to assay H₂O₂ in their simulated Archean atmosphere experiments, as we already did in Ref. [2]. This concerns the leuco crystal violet (LCV) method. The determination of H₂O₂ by LCV (4-[bis [4-(dimethylamino)phenyl]methyl]-N,N-dimethylaniline) and horseradish peroxidase was pioneered by Mottola et al. [5]. It is based on the oxidation of horseradish peroxidase to Compound I by H₂O₂; Compound I then oxidizes LCV to what is commonly abbreviated as CV⁺. The concentration of the intensely blue CV⁺ is monitored at 590 nm [5]. He et al. [6,7] and Wu et al. [8] cite Cohn et al. [9] for this method. We criticized the presence of EDTA during the assay [2]. Wu et al. [1] now claim that “He et al. [12–14] clarified that EDTA was not used in their experiments”. However, none of the three cited papers contains the term “EDTA”. We note that the conditions of the assay with respect to concentrations and pH were changed by He et al. [6,7] and Wu et al. [8], relative to those optimized by Mottola et al. [5]. They should have carried out control experiments, such as assays based on different chemistry, spiking of samples with H₂O₂ as we mentioned [2], and addition of catalase. Furthermore Wu et al. claim that horseradish peroxidase catalytically splits H₂O₂ into 2 HO^•, in contrast to the established reaction mechanisms of peroxidases. Had hydroxyl radicals been formed, a major portion of the LCV would have been hydroxylated.

As we [2] wrote, “ESR evidence does show that there are radicals present in quartz.” However, that dangling silicate bonds generate HO^• and H₂O₂ is a substantial claim that requires stringent proof that has not yet fully been provided. We conclude that, if organisms were exposed to weakly oxidized microenvironments during the Archean, “whiffs of oxygen” [10] would have been responsible, and H₂O₂ generated by minerals on the Archean terrestrial surface is an interesting topic for further research that should take into account that river sand - quartz - accelerates the decay of H₂O₂ [11]. Wu et al. [1] do not contest our conclusion [2] that early organisms in the Archean ocean were exposed to H₂O₂ only after the arrival of oxygenic photosynthesis.

Funding

Not applicable.

CRediT authorship contribution statement

Willem H. Koppenol: Writing – review & editing. Helmut Sies: Writing – review & editing.

Declaration of competing interest

There is no conflict of interest.

Data availability

No data was used for the research described in the article.