NADH and NADPH peroxidases as antioxidant defense mechanisms in intestinal sulfate-reducing bacteria

Animal and human feces typically include intestinal sulfate-reducing bacteria (SRB). Hydrogen sulfide and acetate are the end products of their dissimilatory sulfate reduction and may create a synergistic effect. Here, we report NADH and NADPH peroxidase activities from intestinal SRB Desulfomicrobium orale and Desulfovibrio piger. We sought to compare enzymatic activities under the influence of various temperature and pH regimes, as well as to carry out kinetic analyses of enzymatic reaction rates, maximum amounts of the reaction product, reaction times, maximum rates of the enzyme reactions, and Michaelis constants in cell-free extracts of intestinal SRB, D. piger Vib-7, and D. orale Rod-9, collected from exponential and stationary growth phases. The optimal temperature (35 °C) and pH (7.0) for both enzyme’s activity were determined. The difference in trends of Michaelis constants (Km) during exponential and stationary phases are noticeable between D. piger Vib-7 and D. orale Rod-9; D. orale Rod-9 showed much higher Km (the exception is NADH peroxidase of D. piger Vib-7: 1.42 ± 0.11 mM) during the both monitored phases. Studies of the NADH and NADPH peroxidases—as putative antioxidant defense systems of intestinal SRB and detailed data on the kinetic properties of this enzyme, as expressed by the decomposition of hydrogen peroxide—could be important for clarifying evolutionary mechanisms of antioxidant defense systems, their etiological role in the process of dissimilatory sulfate reduction, and their possible role in the development of bowel diseases.


Materials and methods
Bacterial culture and cell-free extracts preparation.The sulfate-reducing bacteria Desulfovibrio piger Vib-7 (GenBank: KT881309.1)and Desulfomicrobium orale Rod-9 (GenBank: MF939896), which were isolated from feces of a healthy human's large intestine and identified using the sequence analysis of the 16S rRNA gene, were the focus of this study 20,39 .
In a modified liquid Postgate's anoxic medium C, both bacteria were grown 20,[39][40][41] .In order to create an O 2 -free medium, the medium was boiled in water for 30 min before being cooled to 30 °C.Na 2 S (1%) in 1% NaHCO 3 was employed as a reducer prior to the seeding of bacteria in the media (E 0 of the medium was -200 mV).The potential was measured with a pH meter: Greisinger G1500.A sterile 10 N solution of NaOH was used to bring the medium's final pH down to 7.5.The proportion of the inoculate volume to the medium volume was 5%.The bacteria were cultivated anaerobically in flasks for 72 h at 37 °C41 .In addition, reduced FeS and Na 2 S were included in the medium which provided the necessary redox conditions for SRB cultures.Biomass of bacterial cultures was determined photometrically, according to the method previously described in the published paper 39 : about 1 mL of liquid medium without Mohr's salt was transferred into a plastic cuvette and taken to a biophotometer (Eppendorf BioPhotometerD30) for taring.Subsequently, 1 mL of bacterial suspension was transferred into another cuvette and taken again to the biophotometer.Before SRB were used for the experiments, optical density (OD 340 ) was always measured.The optical density of the inoculate was measured and normalized to be the same amount of cells inoculating all bacterial growth.
After 18 h of exponential growth and 36 h of stationary growth, cells were harvested, centrifuged for 30 min at 5000×g at 4 °C, washed with 0.02 M Tris-HCl buffer, and then suspended in the same buffer at a ratio of 2-2.5 g wet weight to 10 mL of the same solution.Bacterial cells were broken and homogenized using the ultrasonic disintegrator at 22 kHz for 1 min at 0 °C with subsequent centrifugation (17,000×g, 60 min, 4 °C) to remove the cell debris and to obtain cell-free extracts.
Assays for enzyme activity and kinetic studies.The rate of NADH or NADPH peroxidation was measured at 340 nm (ɛ = 6220 M −1 cm −1 ).The addition of 2 mM H 2 O 2 started the process.The assay mixture contained cell-free extracts extracts diluted in Tris-HCl buffer (50 mM, pH 7.6) (final volume, 1 mL).As previously mentioned 38 , NADH and NADPH were added in quantities ranging from 50 to 500 μM.The control samples were the same mixture, but without bacterial cell-free extracts.The Bradford method was used to calculate the protein content in the cell-free extracts 42 .
A product proportional to the amount of peroxidase activity is produced when H 2 O 2 and the probe react, and this reaction can be seen using colorimetric (570 nm) and fluorometric (λ ex = 535/λ em = 587 nm) techniques (Sigma-Aldrich kit for the determination of peroxidase activity was used).The amount of peroxidase that lowers 1.0 mol of H 2 O 2 per min at 37 °C is known as one unit (U = 1 μmol/min).As U × mg −1 protein, specific enzyme activity was expressed.Specific enzyme's activity in cell-free extracts of both bacterial strains were also determined under the influence of two important factor pH values (4.0-10.0)and temperature ranges (20, 25, 30, 35, 40, and 45 °C).Under the various settings (pH and temperature), the composition of the same combination remained essentially the same.pH was adjusted with acid (HCl) and base (NaOH) addition.The temperature of the mixture was also adjusted and cultivated under different temperature regimes.
In a standard incubation medium (discussed above) with adjusted physical and chemical properties of the relevant test parameters (i.e., the incubation duration, substrate concentration, temperature, and pH), kinetic study of the enzyme reaction was carried out.The initial (instantaneous) reaction velocity (V 0 ), maximum reaction velocity (V max ), the maximum amount of the reaction product (P max ), and the characteristic reaction time (time of half saturation, τ) were identified as the kinetic parameters describing the H 2 O 2 reaction.
Stoichiometric calculations were used to determine the reaction product's volume.A Lineweaver-Burk plot 43 was used to establish the kinetic parameters that define peroxidase processes, including the Michaelis constant (K m ) and maximal reaction velocity of the substrate degradation.Initial velocities with various substrate concentrations were obtained under normal assay conditions in order to analyze the substrate kinetic mechanism of peroxidases.
To model the kinetic data for quick equilibrium rate equations, the obtained data were also examined by global curve fitting in SigmaPlot (Systat Software, Inc., https:// systa tsoft ware.com/ produ cts/ sigma plot/ sigma plot-how-to-cite-sigma plot/, Palo Alto, CA 94303, the USA) describing ordered sequential ) kinetic mechanisms, where V is the initial velocity, V max is the maximum velocity, K A and K B are the K m values for substrates A and B, respectively, and α is the interaction factor if the binding of one substrate changes the dissociation constant for the other 44 .

Statistical analysis.
The MS Office and Origin (www.origi nlab.com) softwares were used to calculate the data' kinetic and statistical properties.The Student t-test was used to analyze the research findings utilizing methods of variation statistics.The method of least squares was used to determine the linear equation.The absolute value of the correlation coefficient r was ranging from 0.90 to 0.98.The significance of the calculated parameters of the line was tested by the Fisher's F-test.The approximation was deemed accurate approximation when P ≤ 0.05 45 .Overall differences between enzyme activities and kinetic parameters of NADH and NADPH peroxidases were determined by principal component analysis (PCA).Eigenvalue was estimated for two groups to be above zero, explaining 99% of total variance.
Ethics approval and consent to participate.Authors are confirming that all methods were carried out in accordance with relevant guidelines and regulations.Authors are confirming that all experimental protocols were approved by Masaryk University committee (No.EKV-2021-060).Authors are confirming that informed consent was obtained from all subjects and/or their legal guardian(s).

Results
The intestinal SRB D. piger Vib-7 reached the growth phase after 36 h in the stationary phase, but D. orale Rod-9 had the maximum after 60 h.The sample collection was done from both cultures after 24 h (the exponential phase) and 60 h (the stationary phase) of the cultivation (Fig. 1A).Specific peroxidase activity-which is an important enzyme in the process of antioxidant defense in SRB-was measured in the cell free extract of D. piger Vib-7 and D. orale Rod-9 cells, taken during the exponential and stationary phase (Fig. 1B).The highest activity of NADH peroxidase (79.25 and 48.55 U × mg −1 of protein) was detected among extracts of D. piger Vib-7 and D. orale Rod-9, cultivated during the stationary phases.Although, the activity of D. orale Rod-9 were slightly different between the exponential and stationary phases, the activity of NADPH peroxidases in D. piger Vib-7 was similar (p > 0.05) in the exponential and stationary phases.
Enzymatic activity is heavily influenced by environmental and physical factors, including temperature and pH.In some cases, temperature and pH may simulate the activity and at the same time sometimes dissimulate the activity too.The activities of NADH and NADPH peroxidases in intestinal SRB genus Desulfovibrio and Desulfomicrobium have never been monitored with scientific accuracy; the effects of temperature and pH of the reaction mixture on peroxidase activity in the cell-free extracts of the SRB was studied (Fig. 2).
The optimal temperature for enzymatic reactions was determined in both bacterial species of Desulfovibrio and Desulfomicrobium genera and for both enzymes (NADH and NADPH peroxidases) at 35 °C.The increase (max 45 °C) or decrease (min 20 °C) of the optimal temperature resulted in extreme decrease of enzymatic activity in extracts collected during exponential and stationary growth phases.Though, both enzymes showed lower activity at lower temperatures (20-25 °C), during the stationary phase of both tested bacterial cultures, in comparison with results obtained at the optimal temperature (Fig. 2A).The highest enzymatic activity of NADH and NADPH peroxidases for D. piger Vib-7 and D. orale Rod-9 was measured at pH 6.5 to 7.0.for both enzymes Vol:.( 1234567890 during the stationary and exponential phases.The increase of pH resulted in another extreme reduction of enzymatic activity, where the lowest enzymatic activity was at pH 8, while enzymatic activity of D. orale was under the detection limit in the exponential growth phase.A similar trend was observable under the pH conditions lower than 5, where the enzymatic activity was not detectable during the exponential phase in both bacterial strains (Fig. 2B).Obviously, enzymatic activity was inhibited by pH under 5, but in the stationary phase, NADH peroxidase activity was still detectable in very low concentrations (in both bacterial extracts).Thus, as a function of temperature and pH, enzyme activity often displayed bell-shaped curves.These enzymes functioned the best at 35 °C and a pH of 6.5-7.0,respectively.The enzymatic activity in the cell-free bacterial extracts of the SRB was reduced in response to changes in temperature and pH.
Investigation of the kinetic characteristics of both types of peroxidases involved the studying of the reaction dynamics product accumulation (Fig. 3).According to experimental evidence, peroxidase activity kinetic curves tend to saturate (Fig. 3A).According to the analysis of the data, the kinetics of peroxidase activity in the sulfate-reducing bacteria was consistent with a zero-order reaction between 0 and 1 min (the graph showing the relationship between product formation and incubation time was nearly linear throughout this time interval).
Therefore, the incubation time for bacterial cell extracts was 2.5 min.In the entire time range during the exponential development phase, D. piger Vib-7 cell-free extracts produced more NADH and NADPH peroxidase products than D. orale Rod-9 (38 ± 3.52 and 27 ± 2.33 µmol × mg −1 protein) (Table 1).When compared to the NADPH peroxidases of the two extracts, NADH peroxidase activity was higher during the first 1.5 min in the stationary phase of both D. piger Vib-7 and D. orale Rod-9 (Fig. 3A).
By linearizing the data in the P/t; P coordinates, the fundamental kinetic characteristics of the reaction in the sulfate-reducing bacteria were calculated.The concentration of substrate affects how the NADH and NADPH peroxidase activities behave in a kinetic manner (H 2 O 2 ).The examined enzyme's activity rose monotonically as H 2 O 2 concentrations increased from 0.5 to 5 mM, and it remained at the same level (plateau) at substrate concentrations greater than 2.0 mM (Fig. 3B).
The enzyme was saturated with the substrate, and greater doses of H 2 O 2 (2.0-5.0 mM) had no effect on its activity; it remained constant (plateau).The tangent slope and position at where the dependence 1/V; 1/[S] curves intersect the vertical axis were used to identify them.By linearizing the data in the Lineweaver-Burk plot, the fundamental kinetic parameters of NADH and NADPH peroxidase activity in D. piger Vib-7 and D. orale Rod-9 were determined.Both strains had considerably different NADH and NADPH peroxidase kinetic parameters: D. orale Rod-9 and D. piger Vib-7.The maximum amount of the production reaction (P max ) was used to calculate the enzymes' initial (instantaneous) reaction velocity (V 0 ).
As shown in Table 1, V 0 for for NADPH peroxidase reaction was higher in both strains during exponential growth phase in comparison with NADH: D. piger Vib-7 (289 ± 27.43 µmol × min −1 × mg −1 protein) and D. orale Rod-9 (144 ± 13.55 µmol × min −1 × mg −1 protein).Oppositely, the parameter V 0 for NADH peroxidase reaction was more than twice and triplicate higher than NADPH during stationary phase for the both included strains, D. piger Vib-7 and D. orale Rod-9, respectively.The same observation was seen with product reaction (P max ), both during exponential and stationary growth; D. piger Vib-7 had higher NADH and NADPH peroxidase reaction in comparison with D. orale Rod-9.The values of the reaction time (τ) during the exponential growth phase was higher (NADH peroxidase reaction, 0.36 ± 0.29 min) for D. piger Vib-7, but NADPH was higher for D. orale Rod-9 (0.19 ± 0.017 min).D. orale Rod-9 had higher the values of the reaction time (τ) for both peroxidase reactions (NADH and NADPH) during the stationary phase in comparison with another tested strain (D. piger Vib-7).V max values presented with the same tendency, as NADH values were higher in D. piger Vib-7 strain, but NADPH for D. orale Rod-9 strain; ahough, the differences during the stationary phase are almost neglectable.
Michaelis constant (K m ) was lower during the exponential phase for D. piger Vib-7 strain, the both NADH and NADPH.

Discussion
Antioxidant enzymes are endogenous systems can scavenge ROS 30 .SOD, catalase, and peroxidases belong to antioxidant enzymes that are able to prevent intracellular ROS formation, same as lipid peroxidation 46 .While catalase breaks down superoxide radicals into water and O 2 , superoxide dismutase is able to convert superoxide radicals into H 2 O 2 28 .Heme-containing enzymes called peroxidases (EC 1.11.1.7;hydrogen peroxide oxidoreductase) can catalyze the oxidation of substrates utilizing H 2 O 2 47 , a non-radical ROS product of regular aerobic metabolism.The high amount of H 2 O 2 could be converted into more reactive ROS (e.g., hydroxyl radicals capable of oxidizing biomolecules) that can damage tissue, cause cell death or carcinogenesis, aging and have an effect on cardiovascular diseases.Therefore, a crucial component of overall antioxidant activity is H 2 O 2 scavenging activity 48,49 .
Table 1.Kinetic parameters of the NADH and NADPH peroxidases from intestinal sulfate-reducing bacteria* *V 0 is initial (instantaneous) reaction rate; P max is maximum amount (plateau) of the reaction product; τ is the reaction time (half saturation period), V max is maximum rate of the enzyme reaction; K m is Michaelis constant which was determined by substrate (hydrogen peroxide).Statistical significance of the values mean ± SD, n = 5; ***P < 0.001, compared to the D. piger Vib-7 strain.The discovery of new species of SRB resulted in significant changes in their taxonomy, the same as in concepts about SRB metabolism.Nowadays, many SRB are considered to be aerotolerant anaerobes, and O 2 might play an important physiological role in the metabolism of SRB.It is suggested that SRB use O 2 as a final acceptor of electrons during the synthesis of ATP 50,51 .It should be also noted that O 2 inhibits some of the critical enzymes of SRB, like lactate dehydrogenase and NAD dependent alcohol dehydrogenase 52 .
Some SRB are partially aerotolerant to some degree [53][54][55][56] , and even after prolonged exposure to O 2 , some SRB species can resume anoxic growth.These aerotolerant SRB mainly contain SOD and catalase 55,57,58 .There is not much information gained by previous experiments about enzymes involved in O 2 consumption by SRB.The O 2 reduction chain consisting of an NADH oxidase (NADH rubredoxin oxidoreductase), which is present in D. gigas 59,60 .NADH oxidase activities have also been found in D. desulfuricans NCIB 8301 57 and D. vulgaris Hildenborough 60 .Hardy and Hamilton 55 found O 2 reduction activities in several D. vulgaris, though NADH oxidase activity was not detected.According to the previous work, it was observed that O 2 reduction in SRB species takes places in the periplasm and is linked to cytochrome c 3 , as Postgate 61 proposed for D. desulfuricans strain.Probably at least three independent systems are able to reduce O 2 in D. desulfuricans CSN.Out of those three systems, one system is active only at low O 2 concentrations, but it is inhibited by high O 2 concentrations; the rest two systems are active at all O 2 concentrations 38 .
The suggestion that the former system is probably active in the periplasm, and it is linked to cytochrome c 3 .Oxidation activities of NADH and NADPH are in soluble cell extracts at all O 2 concentrations and these activities declined with a decrease of O 2 concentration.NADH oxidase activity was present in all strains that were examined, they all responded similarly to changes in O 2 concentrations 32,61 .
These results suggest that the SRB contain NADH oxidase that able to reduce O 2 directly to H 2 O, though some SRB strains can reduce O 2 to H 2 O 2 ; H 2 O 2 is further reduced to H 2 O, as these strains possess the NADH peroxidase.Examined D. desulfuricans strains contained NADH and NADPH oxidases and peroxidases 38 , therefore these findings correspond with our research that included D. piger Vib-7 and D. orale Rod-9 where the activity of these enzymes were also observed.
In the research by van Niel et al., these strains do not accumulate polyglucose during growth with various substrates.These properties distinguish them from the following strains: D. salexigens and D. gigas.In all cases, the addition of substrates resulted in a rapid decline of NADH oxidase activity.The same observation was seen with NADPH oxidase, NADH peroxidase, and NADPH peroxidase, same as with O 2 consumption by the whole cells of D. desulfuricans strains.It is possible that reactive intermediates-which damage the enzyme-are formed during oxidation reactions with O 2 .The more extensive will be the damage and thus inactivation when the faster oxidation is taking place 38 .
The facultative anaerobic bacterium Lactobacillus casei IGM394 strains carry several genes that are allowing the strain to tolerate O 2 and reactive oxygen species (ROS), though the complete functions have not been revealed yet.The decreased growth and high H 2 O 2 accumulation were observed in the NADH peroxidase in comparison with wild types.Due to the H 2 O 2 degradation capacity, it was revealed that NADH peroxidase is a major H 2 O 2 degrading enzyme in L. casei IGM394.Conversely, the H 2 O 2 tolerance mechanism is dependent only on NADH peroxidase in L. casei IGM394 62 .
The inactivation in this organism is considered to be an intrinsic property of the enzymes and their substrates.Inactivation only happens in the case when both oxidizable substrates and O 2 are present.According to the results, the enzyme-substrate complex is prone to denaturation.Enterococcus faecalis NADH oxidases are one of the best studied.D. gigas 60 and E. faecalis 63 NADH oxidases both contain FAD, no cental metal ions and they are sensitive to sulfhydryl agents.The gradual removal of FAD is the reason for the instability of S. faecalis NADH oxidase 64 .Although the addition of FAD to the cell extracts increased the rate of oxidation of NADH, but it had no effect on the inactivation rate.The experiments were conducted also on the mutant strain where NADH peroxidase activity was observed, but not in the wild type.NADH peroxidase activities in in vitro systems containing NADH hydrogen peroxide, and a bacterial NADH oxidoreductase from Desulfovibrio vulgaris and from Clostridium perfringens was also shown 65 .
Desulfovibrio species (SRB group) are ubiquitous anaerobic microorganisms and they have a large metabolic diversity 4,18,22,[66][67][68] .All SRB members are unified because they use sulfate as the terminal electron acceptor, reducing it to H 2 S. Though, SRB species are classified as strict anaerobes, they are able to deal with the temporary presence of O 2 (in natural habitats, such as marine surface waters, microbial mats, sewers, rice paddies and oil pipelines), and several Desulfovibrio species was found to be able to oxidize organic substrates under low levels of O 2 69 .Otherwise, Desulfovibrio that are aerotolerant cannot utilize O 2 for growth 50 , therefore, in the presence of sulfide, SRB are more O 2 sensitive 53 .
To obtain more insights with regard to peroxidases in SRB, we examined the genomes of closely-related organisms.However, despite our efforts to ascertain more concrete information, the findings are the following.Only one refseq genome for Desulfomicrobium orale (https:// www.ncbi.nlm.nih.gov/ nucco re/ NZ_ CP014 230.1) is available at NCBI.Desulfomicrobium orale genes were found to encode for a putative Dyp-type peroxidase (WP_066605144.1), a putative thiol-peroxidase (WP_066607718.1), a putative superoxide dismutase (WP_066603707.1), and two putative peroxireducins (WP_066605219.1;WP_066606413.1).However, which is more relevant to our study, several oxidoreductases are encoded in the genome, such as two putative FADdependent oxidoreductases (WP_066608725.1;WP_066606935.1;WP_066605517.1),two NAD(P)/FADdependent oxidoreductases (WP_066604797.1;WP_151192312.1),and two NAD(P)-dependent oxidoreductases (WP_066606986.1;WP_066607102.1).From our perspective, it might be reasonable to consider that the last five enzymes could play a role in the specific enzyme activities under investigation.Nevertheless, the enzyme traits predicted by the automated genome annotation pipeline might not necessarily suggest a direct involvement of these enzymes in peroxidase functions.Identifying the enzyme characteristics would entail the cloning, expression, and characterization of each of the presumed enzymes.Alternatively, another approach could involve pinpointing the pertinent putative peroxidases through transcriptomics within specific growth experiments.Both routes would be interesting to follow in future studies.Still, our study does not primarily concern the genetic properties of the examined enzymes through a bioinformatics approach.There is also a genome of a closely related Desulfovibrio piger available at NCBI.This genome is not a refseq genome.While we made an effort to locate the information, we did not explore the automated annotation pipeline results for this genome.Moreover, we conducted an additional literature search to determine whether peroxidase activities had previously been reported for other SRB.Regrettably, no records pertaining to this aspect were found.Hence, the activity of NADH and NADPH peroxidases has not been tested till now, too.There is an overall scarcity about this information on peroxidases in environmental SRB.Regarding environmental SRB, the presence of peroxidases might be contingent upon the specific strain or species under investigation.Some SRB may possess peroxidases as part of their metabolic pathways, while others may rely on alternative enzymes or mechanisms for managing oxidative stress.

Conclusions
NADH and NADPH peroxidases are important enzymes that are putatively involved in the antioxidant defense systems of intestinal SRB.Both peroxidases could represent an evolutionary response to oxidative stress, and they can sustain their activity over a broad range of temperature and pH conditions, support the process of dissimilatory sulfate reduction, and the production of H 2 S. At 35 °C and pH 7.0, the maximal specific enzymatic activities were found.
In the case of D. piger Vib-7 compared to D. orale Rod-9, the kinetic parameters of enzyme specific activity, including V 0 and V max , were significantly higher.The concentrations of the substrate (H 2 O 2 ) affected the kinetic parameters of enzyme reactions.Between D. piger Vib-7 and D. orale Rod-9, the K m values were notably different throughout the exponential and stationary growth phases.The NADH peroxidase of D. piger Vib-7 is the exception to this finding.D. orale Rod-9 displayed much higher K m during both phases.
As D. piger Vib-7 and D. orale Rod-9 peroxidases could be involved in their respective metabolisms as well as in the dissimilatory sulfate reduction and the production of H 2 S, the results obtained may be considered as significant insights and perspectives for clarification of the etiological role in the development of bowel diseases in humans and animals. https://doi.org/10.1038/s41598-023-41185-3

Figure 1 .
Figure 1.Biomass accumulation of D. piger Vib-7 and D. orale Rod-9 (A) as well as NADH/NADPH peroxidase activity (B) in cell-free extract of intestinal SRB depending on their physiological phase (mean ± SD, n = 5).

Figure 2 .Figure 3 .
Figure 2. The effect of temperature (A) and pH (B) on NADH and NADPH peroxidase activity (mean ± SD, n = 5) in the cell-free extracts prepared from various bacterial growth phases (exponential and stationary).

Figure 4 .
Figure 4. Principal component analysis (PCA) based on kinetic parameters of NADH and NADPH peroxidase activity in the cell-free extracts of D. piger Vib-7 (Dv) and D. orale Rod-9 (Dm) prepared from various bacterial growth phases (exponential (EXP) and stationary (Sta)).