Investigation of the Ferredoxin’s Influence on the Anaerobic and Aerobic, Enzymatic H2 Production

Ferredoxins are metalloproteins that deliver electrons to several redox partners, including [FeFe] hydrogenases that are potentially a component of biological H2 production technologies. Reduced ferredoxins can also lose electrons to molecular oxygen, which may lower the availability of electrons for cellular or synthetic reactions. Ferredoxins thus play a key role in diverse kinds of redox biochemistry, especially the enzymatic H2 production catalyzed by [FeFe] hydrogenases. We investigated how the yield of anaerobic and aerobic H2 production vary among the four different types of ferredoxins that are used to deliver electrons extracted from NADPH within the synthetic, fermentative pathway. We also assessed the electron loss due to O2 reduction by reduced ferredoxins within the pathway, for which the difference was as high as five-fold. Our findings provide valuable insights for further improving biological H2 production technologies and can also facilitate elucidation of mechanisms governing interactions between Fe–S cluster(s) and molecular oxygen.


INTRODUCTION
Ferredoxins (Fd) are redox proteins that mediate electron metabolism in numerous kinds of cells across diverse organisms. The [Fe-S] cluster(s) in ferredoxins are responsible for electron receipt and transfer to redox partners. The type and number of [Fe-S] clusters in a ferredoxin can vary, which in turn affect the rate at which electrons are delivered, as well as redox potential of the protein (Guan et al., 2018). For example, Synechocystis sp. PCC 6803 ferredoxin (SynFd) has one [2Fe-2S] cluster while Clostridium pasteurianum ferredoxin (CpFd) harbors two [4Fe-4S] clusters. The midpoint redox potentials for the two are −412 and −387 mV, respectively (Bottin and Lagoutte, 1992;Brereton et al., 1999).
One of ferredoxins' redox partners is a metalloprotein called hydrogenase. Hydrogenases can receive electrons from reduced ferredoxins (Fd red ) and combine them with protons to produce H 2 . The enzymatic activity of hydrogenases, especially the [FeFe] subtype proficient in H 2 production such as the one from the C. pasteurianum (CpI), can be exploited for biological H 2 production (Lu and Koo, 2019); wide deployment of the technology can contribute to reduction of CO 2 emission while supplying H 2 . Toward this aim, researchers have successfully developed both fermentative and photosynthetic pathways for reducing ferredoxins, which can subsequently be used for the enzymatic H 2 production (Smith et al., 2011;Yacoby et al., 2011). To date, however, people have tested H 2 production from these pathways only in an anaerobic reactor due to the high O 2 sensitivity of [FeFe] hydrogenases.
O 2 is a byproduct of photosynthesis and also an effective reagent for regenerating adenosine triphosphate (ATP) during fermentation. As such, it is an unavoidable constituent of the aforementioned biological H 2 production processes; its influence on electron flow and H 2 production need to be understood. Previous work showed that Fd red is capable of reducing O 2 into superoxide and peroxide (Allen, 1975), which is known as the Mehler reaction within the photosynthetic pathways. Hosein and Palmer also reported the Fd oxidation by O 2 and its autocatalytic nature (Hosein and Palmer, 1983). Since Fd red is the source of electrons for the enzymatic H 2 production, presence of O 2 results in a lower H 2 yield via decreasing the amount of electrons delivered to the hydrogenases (Benemann et al., 1973;Koo and Swartz, 2018). O 2 also drives inactivation of the hydrogenases, which is irreversible and fast (within minutes under the atmospheric [O 2 ]) for most [FeFe] kinds (Lu and Koo, 2019). By fusing ferredoxin to an [FeFe] hydrogenase, Eilenberg et al. (2016) successfully increased the photosynthetic H 2 production in the presence of O 2 , proposing that Fd red delayed inactivation of the hydrogenase at the expense of electrons. The authors also confirmed the increase in aerobic H 2 production by fusing different types of ferredoxin and hydrogenase (Koo, 2020).
We developed physiological assays and analytical methods for characterizing O 2 sensitivity of [FeFe] hydrogenases during H 2 production (Koo et al., 2016). Using this method, the authors studied the enzymatic H 2 production in the presence of O 2 at a greater detail to better understand its implications for biological H 2 production and photosynthesis. First, we used isotopically labeled O 2 , ( 18 O 2 ) to confirm O 2 reduction by Fd red . We then analyzed how the rates of O 2 reduction vary with varying concentrations of reactants, as well as with different types of ferredoxins harboring different kind and number of Fe-S clusters. Lastly, we studied how electron loss from Fd red to O 2 varies among different combinations of ferredoxin NADP + reductases (FNR) and ferredoxins with the goal of identifying the most effective combination in minimizing the electron leakage during the enzymatic H 2 production.

O 2 Reduction and Aerobic H 2 Production Measurements
The O 2 reduction experiments were conducted in 8.4 mL crimp vials where 840 µL reaction mixtures contained the following (unless stated otherwise): 50 mM Tris buffer pH 7.0, 10 mM G6P, 4 units of G6PD, 5.0 mM NADPH, 5.0 or 50 µM FNR, and 5.0 µM of Fd. The reaction mixtures were prepared inside a N 2 -only glovebox (CHOA Engineering). Before sealing the vials with rubber septa, magnetic stir bars were added for mixing. After removal from the glovebox, the sealed vials were placed on a stir plate to initiate mixing at 300 rpm. O 2 was introduced to the headspace at t = 0 min (or 10 min when examining various pairs of FNR and Fd) by using a syringe with a 20-gage needle (Daehan Sciences); air or a gas-tight handheld tank filled with O 2 was used as a source. O 2 and H 2 concentrations were measured by sampling 200 µL of the headspace with a valved 23gage needle (Daehan Sciences), and using gas chromatography (GC 6500, YL Instrument). The H 2 production experiments were done in the same manner with the addition of 10 nM CpI to the aforementioned reaction mixtures inside the anaerobic glovebox.

O 2 Reduction Experiments
The amount of H 2 18 O formed by the reaction mixtures (50 mM Tris buffer pH 7.0, 10 mM G6P, 4 units of G6PD, 5.0 mM NADPH, 5.0 or 50 µM FNR, 5.0 µM of Fd, 4.2 U SOD and 4.2 U catalase) were measured as follows. The 8.4 mL crimp vials (Thermofisher Scientific) were first opened using a decapper, and the reaction buffer were immediately put in the 80 • C water bath (Daehan Sciences) for the following hour to terminate any ongoing enzymatic reactions. Next, the reaction buffer was distilled at 120 • C and only the water vapor was collected by condensation. The collected samples were mixed with distilled H 2 O at two different ratios (2-and 10-fold dilution) and submitted together with a sample containing only distilled H 2 O to the mass spectrometry (National Center for Inter-University Research Facilities, Seoul) for analysis of H 2 18 O content.

Confirmation of 18 O 2 Reduction by Fd red
We have previously reported O 2 consumption by SynFd red during fermentative H 2 production where electrons are extracted from NADPH and delivered to hydrogenases via RrFNR and SynFd (Koo et al., 2016). In this study, we replaced the source of O 2 from air to 10 vol% 18 O 2 . This allowed us to directly measure accumulation of H 2 18 O in the buffer using mass spectrometry. Since Fd red was reported to be capable of only uni-and divalent reduction of O 2 (Allen, 1975), we modified the biochemical reaction as described in Figure 1. Instead of adding the hydrogenase enzyme for the NADPH-driven H 2 production (blue arrows from Fd red ), SOD and catalase were added in the absence of the hydrogenase (red arrows from Fd red ) to increase the likelihood that any reactive oxygen species (ROS) generated by Fd red would be converted into H 2 18 O for isotopic detection. Four reaction mixtures were prepared, incubated with 10 vol% 18 O 2 for an hour, and analyzed to determine (1)   addition of reagents in reaction sequence (Figure 1) starting at SynFd clearly confirmed our previous finding (Koo et al., 2016): SynFd red is responsible for O 2 consumption (Figure 2). The results also suggested that SynFd red is not capable of reducing O 2 fully into H 2 O. This was evident from the observation that a significant amount of H 2 18 O was formed only in the presence of both SOD and catalase. The fact that Synechocystis sp. cells have native SOD and catalase is consistent with our interpretation in that these enzymes can rapidly remove harmful O 2 radicals generated by SynFd red .
Assuming that SOD do not impact the O 2 reduction activity of SynFd red , we expected to see the decrease in headspace O 2 reduce by half (Figure 1, following the red arrows only from Fd red ) in the mixture containing only up to SOD. This is indeed what we observed: The loss of headspace O 2 after an hour decreased from roughly 2,900 to 1,600 nmoles upon addition of only SOD, approximately 50% reduction considering experimental error. We also expected to see a further reduction in the amount by which headspace O 2 decreases upon addition of catalase to the mixture containing SOD (Figure 1) since O 2 is regenerated in the presence of both. Indeed, the loss of headspace O 2 after an hour further decreased to roughly 1,000 nmoles. The water molecules containing 18 O were only formed when both SOD and catalase were added to the reaction mixture. Furthermore, the amount of H 2 18 O produced, 1,500 nmoles, is similar to the stoichiometrically expected value of 1,300 nmoles: Each peroxide molecule generated by SOD will be converted into a water molecule by catalase. Based on the headspace O 2 loss, the average turnover number (TON) of O 2 reduction by SynFd was 11 per minute per reduced ferredoxin. It is important to note that this TON is not physiologically relevant. Fd mostly delivers electrons for NADPH regeneration inside cells while, in our experiments, the SynFd molecules were reduced using the electrons extracted from NADPH to consume O 2 .

Kinetic Study on O 2 Reduction by Fd red
We next examined how the rate of electron loss to O 2 varies with respect to O 2 partial pressure in the headspace. As discussed in the previous section, SOD and catalase affect the apparent O 2 consumption by regenerating about half of the putative O 2 radicals back to O 2 . We thus conducted the following experiments in the absence of SOD and catalase. The concentrations of reagents inside the sealed glass vials were as usual except for RrFNR, which was increased by 10-fold to 50 µM RrFNR. The higher [RrFNR] was chosen in order to enhance electron flux to Fd (K M of 18 or 39 µM for SynFd and CpFd, respectively) and thereby increase changes in the O 2 peaks for more accurate analysis.
The results summarized in Figure 3 and Supplementary  Figure 1 indicated that the O 2 reduction rate, or equivalently the electron loss rate is directly proportional to the headspace [O 2 ]. A linear relationship between the two was observed for both types of Fd from 1 to 21 vol% O 2 in the headspace of the reactor vial. Within this range, the observed O 2 consumption rate varied from 21 to 255 or 46 to 479 nmoles/min for SynFd and CpFd, respectively. The corresponding specific rate of electron loss to O 2 ranged between 5 and 61 per minute for SynFd (assuming only univalent reduction of O 2 ) and approximately 1.9-fold higher for CpFd. As expected, increasing the [RrFNR] from 5 to 50 µM increased the specific rate of electron loss, but only by about three-fold.
Based on these observations, we analyzed the kinetic parameters for the reductive O 2 consumption. The following rate law (Eq. 1a) was proposed where the consumption is modeled as a non-enzymatic biochemical reaction between Fd red and O 2 :  (Figure 3), which suggests that the O 2 reduction by Fd red follows a pseudo first order rate law under the experimental conditions. The rate constant of this kinetic model (eqn. 2, b = 1), namely k 1 , is  (Bertini et al., 1995;Van Den Heuvel et al., 2003 (Yao et al., 2012). The midpoint redox potentials (RE) are similar: −412 and −387 mV for SynFd and CpFd, respectively (Grzyb et al., 2018). The dissociation constants with RrFNR and CpI are also not significantly different. In this regard, the difference in the range and nature of oxidation states appear to be more important with respect to the Fd's O 2 reduction activity. The results in the subsequent section suggests that the local environment surrounding the Fe-S clusters also matter.

Aerobic H 2 Production From Various Pairs of FNR and Fd
We have previously reported that the rate of O 2 consumption is different when SynFd versus CpFd is used for the NADPHdriven H 2 production pathway (Koo et al., 2016). The rate was approximately twice as fast with CpFd, meaning that more electrons are lost to O 2 reduction instead of being used for H 2 production by the hydrogenase. The choice of CpFd over SynFd would therefore result in a lower NADPH-driven H 2 production efficiency. In this manner, the choice of Fd can impact the efficiency and overall yield of aerobic, biological H 2 production when Fd molecules deliver electrons to the hydrogenase enzyme.
We examined various combinations of Fd and FNR to study how the electron loss to O 2 differs during H 2 production (Figure 1, co-existence of both the blue and red arrow pathways) under 5.0 vol% O 2 . Different FNRs were evaluated as well since the electron flux rate through the FNR could influence the steady state [Fd red ] (Bingham et al., 2012;Lu et al., 2015), which in turn would affect the rate of electron loss to O 2 . The concentrations of the reagents were as follows: 10 mM G6P, 1 U G6PD, 5 mM NADPH, 5 µM FNR, 5 µM Fd, and 10 nM WT CpI in 50 mM Tris-HCl buffer pH 7.0. The reaction lasted for an hour, and we analyzed the data with respect to (1) anaerobic FNR TON during H 2 production, (2) relative aerobic H 2 yield, and (3) the overall change in the headspace [O 2 ].
The results (Table 1) indicated that electrons in Fd red are lost to O 2 at a faster rate with a higher (anaerobic) FNR TON within the NADPH-driven H 2 production (r = 0.82). This was expected since a higher FNR TON means a greater electron flux to Fd. In contrast, there was insignificant correlation between the anaerobic FNR TON and the relative aerobic H 2 yield of the reaction (r = −0.25). This confirmed our previous finding from the CpI mutagenesis study where we reported insignificant relationship between the two variables (Koo and Swartz, 2018). This observation suggests that it may be possible to improve both the electron flux and aerobic H 2 yield by engineering FNR and/or Fd.  a The relative aerobic H 2 yield refers to the percent of expected production based on the anaerobic production rate. For example, for the combination of SynFNR and SynFd, the anaerobic FNR TON is 5.2 per minutes and the amount of H 2 produced anaerobically over an hour is about 1.3 µmoles; the aerobic H 2 yield of 8.5% means that 0.1 µmoles are produced instead in the presence of 5.0 vol% O 2 in the headspace. For further explanations, please refer to our previous work (Koo et al., 2016). b This is calculated based on the assumption of univalent O 2 reduction by Fd.
In contrast, CpFd lost the most electrons to O 2 among the four types of Fd studied in this experiment. The difference in efficiency between the two ferredoxins was striking: The overall amount of O 2 consumed by CpFd red over an hour was as much as four-fold of the amount by AnFd red . AnFd harbors one [2Fe-2S] cluster with the midpoint RE potential of −405 mV (Jacobson et al., 1993). This is not much different from ZmFd, for example, which also features one [2Fe-2S] cluster and the midpoint RE potential of −390 mV (Shinohara et al., 2017). The difference in terms of electron leakage to O 2 reduction during H 2 production, however, is nearly two-fold. The slightly higher midpoint RE cannot explain this difference since SynFd loses more electrons to O 2 in spite of the lower midpoint RE potential (−412 mV) than AnFd (Table 1).
These results indicate that other factors affect the partitioning of the electron flux between H 2 production and O 2 reduction. Given the substantial difference for the same type of Fd when a different FNR is used, we suspect that the binding dynamics is one of the factors influencing the partitioning. The reported binding affinities between native Fds and FNRs used in this study range between 15 to 50 µM (Lu et al., 2015;Shiigi, 2015). No obvious relationships between these affinities and the FNR TON or electron flux to O 2 reduction were observed, likely owing to the complex nature of multi-component interactions in our assay (Figure 1). A fully exhaustive study on the binding interactions between each pair within the assay may help to elucidate the electron partitioning phenomena.

CONCLUSION
We studied how the choice of ferredoxin affects the aerobic, biological H 2 production catalyzed by the [FeFe] hydrogenase molecules. The results revealed that the electron flux from the reduced ferredoxin can partition between O 2 and the hydrogenase and that the ratio of partitioning can vary among the different types of ferredoxin. We compared the results obtained by combining two different types of FNR with four different types of Fd. The differences in the amount of O 2 reduced and H 2 produced over an hour were as large as 4.7-and 2.3fold of the lowest set. Our findings suggest that it is possible to reduce the electron loss to O 2 by varying Fd and/or FNR within the biological H 2 production pathway. The results also hint the possibility of simultaneously improving the TON of H 2 production (which is limited by FNR TON in the NADPHdriven reaction). Since the two redox proteins are commonly found across many forms of life, large combinatorial libraries may be screened to find even better pair to enhance biological H 2 production beyond the current state of the art.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
JK planned and conducted research. He also wrote the manuscript, analyzed data. YC conducted part of the experiments and also contributed to analysis of some of the data. Both authors contributed to the article and approved the submitted version.

FUNDING
This study was funded by the National Research Foundation of Korea (NRF-2019R1C1C1002642).