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Journal of Bacteriology, March 2008, p. 1584-1587, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01562-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Reinvestigation of the Steady-State Kinetics and Physiological Function of the Soluble NiFe-Hydrogenase I of Pyrococcus furiosus

Daan J. van Haaster, Pedro J. Silva, Peter-Leon Hagedoorn, Jaap A. Jongejan, and Wilfred R. Hagen^*

Department of Biotechnology, Delft University of Technology, The Netherlands

Received 28 September 2007/ Accepted 10 December 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (20 µM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 µM dissolved CO). Since the K_m for NADP⁺ is 37 µM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Hydrogenase catalyzes the reversible conversion of molecular hydrogen into two protons and two electrons (8). Although hydrogenases occur in a wide variety of microorganisms from each of the three domains of life, they all have a structural blueprint in common: the active site encompasses an organometallic cofactor based on a sulfur-bridged (Fe, Fe) or (Fe, Ni) pair of metal ions with CO and CN^– ligands on the Fe, with hydrophobic and hydrophilic channels to the protein's surface for transport of H₂ and protons, and with a [4Fe-4S] cubane for electron transfer (the only exception is the iron-sulfur cluster-free hydrogenase from methanogenic archaea [9]). In FeFe-hydrogenases the cubane is an integral part of the active site via a bridging cysteine sulfur. In NiFe-hydrogenases the cubane is in a separate subunit; however, it is spatially close to the dinuclear center, and it is thus designated a "proximal" cluster. Structures for electron transfer beyond this cubane exhibit considerable variation in hydrogenases having different origins, reflecting different functions in the metabolism of different species (6, 20). Pyrococcus furiosus is a strict anaerobe and a hyperthermophilic marine archaeon that grows optimally at 100°C by saccharolytic fermentation or by S⁰-dependent peptidolytic fermentation (3). P. furiosus has two types of hydrogenases: a coenzyme-dependent heterotetrameric soluble enzyme and a multimeric (14-subunit) transmembrane enzyme. Both hydrogenases are encoded polycistronically. A duplication of the operon for the soluble enzyme encodes a low-activity paralog, designated soluble hydrogenase II, that is expressed significantly during growth on maltose (11). There is also a duplication of the operon for the membrane-bound hydrogenase, and transcription of this operon (13 open reading frames) has been established (21). All P. furiosus hydrogenases are the NiFe type; however, for the second membrane-bound enzyme there is a structural gene in which the codons for the bridging Cys ligands to a putative dinuclear NiFe center are replaced by codons for a glutamate and an aspartate, which would be unprecedented substitutions (17) suggestive of an active site and an activity different from those of the hydrogenase.

The physiological role of the soluble hydrogenase(s) in P. furiosus is not clear; workers have described several reassignments of the putative function. Originally, the enzyme was called a new type of evolution hydrogenase because, in contrast to all known NiFe-hydrogenases, the H₂ evolution activity at 80°C was found to be approximately fourfold higher than the H₂ oxidation activity (7) and sensitivity to inhibition by CO appeared to be absent (1, 2). Ferredoxin was proposed to be the natural electron donor (7). It was subsequently found that the enzyme also has S⁰ reduction activity and could therefore be a bifunctional "sulfhydrogenase" (10). Then it was found that the direct reaction with ferredoxin is artifactual, and the natural electron donor was proposed to be NADPH, which in turn was formed by the action of the enzyme ferredoxin:NADP⁺ oxidoreductase (11, 12). However, the reported midpoint potential values for the iron-sulfur clusters are relatively high for H₂ production (2, 5). Later, it was found that P. furiosus membranes contain a membrane-bound hydrogenase with high H₂ production activity (14), and this seems to eliminate the need for a (lower) soluble H₂ production activity and, therefore, leaves the soluble enzyme without an identified function. In another study of the membrane-bound hydrogenase the ferredoxin was identified as its redox partner (17). It was also determined that the hydrogenase activity was sensitive to the proton translocation inhibitor dicyclohexylcarbodiimide and that the activity of the soluble hydrogenase was not sufficient to explain H₂ production levels in vivo (17). This led to the proposal that proton respiration is coupled to energy transduction in a transmembrane hydrogenase complex (17), which was later verified by observation of ATP production coupled to the production of H₂ in membrane vesicles (13). The soluble hydrogenase was proposed to function as a safety valve for the disposal of excess reducing equivalents and/or as a conversion system that reuses the waste product of respiration, H₂, for the production of NADPH for anabolic metabolism (17). The latter assignment makes the soluble hydrogenase a hyperthermophilic equivalent of conventional NiFe-hydrogenases (i.e., physiologically functioning in H₂ oxidation), and this appears to be inconsistent with the previous reports of high production activity (7) and of a relatively high K_m value for H₂ in the oxidation reaction (11). We therefore reinvestigated the steady-state kinetics of the soluble hydrogenase.

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Growth of the organism and purification of the enzyme. P. furiosus DSM 3638 was routinely grown at 90°C in a 140-liter fermentor with starch as the carbon source, and its soluble NiFe-hydrogenase I was isolated and purified as described previously (17). The protein concentration was determined by the bicinchoninic acid assay (18). The absorbance at 562 nm was determined with bovine serum albumin as the standard.

Enzyme activity assays. Methyl viologen reduction activity was measured optically with a glass fiber charge-coupled device array spectrophotometer (Avantes AVS-S2000) by determining the increase in absorption at 600 nm using an ₆₀₀ value of 9.7 mM^–1 cm^–1 (or for high viologen concentrations the increase in absorption at 500 nm using a ₅₀₀ value of 2.0 mM^–1 cm^–1) at 80°C under anaerobic conditions in a 1-ml cuvette with a rubber stopper. The cuvette was filled with a given concentration of methyl viologen in 100 mM EPPS [4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid] (pH 8.0), and the preparation was saturated with hydrogen by bubbling. The reaction was initiated by addition of enzyme. One unit of activity was defined as the activity required to reduce 2 µmol of methyl viologen to the semiquinone radical per min. In a similar protocol, NADP⁺ reduction activity was measured by determining the increase in absorption at 340 nm (₃₄₀, 6.2 mM^–1 cm^–1). One unit of activity was defined as the activity required for the formation of 1 µmol of NADPH per min.

Hydrogen consumption activity was measured amperometrically under anaerobic conditions with a Clark-type electrode (YSI model 5331; Yellow Springs Instruments, Yellow Springs, OH) polarized at 600 mV versus the Ag/AgCl reference electrode in a 2-ml cell with a water jacket. The temperature was kept at 80°C with circulating water. The dual electrode was interfaced to a personal computer via a National Instruments 16-bit ATD converter, and current data were collected with a LabView program at 0.1-s intervals. The assay mixtures contained 1 mM methyl viologen in 2 ml of 100 mM EPPS (pH 8.0) and a variable concentration of hydrogen obtained by dilution with a buffer solution saturated with hydrogen. The solubility of hydrogen at 80°C is 745 µM (4). One unit of enzyme activity was defined as the activity required for the oxidation of 1 µmol of hydrogen per min.

Hydrogen production activity was measured with the same amperometric setup; however, the 1 mM methyl viologen was prereduced to the semiquinone form by using 5 mM sodium dithionite. Different concentrations of CO were added by injection of specific volumes of a CO-saturated EPPS buffer solution. The solubility of CO at 80°C is 691 µM (4). The reaction was initiated by addition of enzyme. One unit of enzyme activity was defined as the activity required for production of 1 µmol of hydrogen per min.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
In different physiological contexts hydrogenases function either in the consumption or in the production of molecular hydrogen. According to a long-standing paradigm, NiFe-hydrogenases frequently are H₂ uptake enzymes with apparent high affinities for H₂, as reflected by Michaelis constants for H₂ in the micromolar range; in contrast, FeFe-hydrogenases function mainly as H₂ production catalysts with K_m values for H₂ in the hundreds of micromolar range (8, 19). A second criterion for distinguishing the functions of these enzymes is the ratio of uptake activity to production activity of purified enzymes, which is typically 100 to 1,000 for NiFe-hydrogenases and ca. 10 for FeFe-hydrogenases (8). Therefore, it was a complete surprise when the first description of a soluble NiFe-hydrogenase from P. furiosus indicated that the ratio of uptake activity to production activity at 80°C was 0.25 (7). The suggestion that this enzyme could be the first representative of a novel production-type NiFe-hydrogenase (7, 12 ) was later corroborated by the finding of a K_m value for H₂ of 140 µM (11), which is 1 or 2 orders of magnitude greater than typical values for mesophilic NiFe-hydrogenases (19). Recently, however, we approached the steady-state kinetics of hydrogenases from a previously unexplored angle, progress curve analysis (19), and our results for the soluble P. furiosus enzyme suggested that the affinity for hydrogen is much higher, as evident from an apparent K_0.5 value for H₂ of 10^–5 M, which is 1 order of magnitude less than the previously reported K_m value.

To resolve this apparent inconsistency, we carried out a Michaelis-Menten analysis of the reaction, and the results are summarized in Fig. 1. The experiment was done under conditions (80°C, 100 mM EPPS buffer [pH 8]) identical to those employed in previous work by Ma et al. (11), except that the substrate H₂, for which the K_m was to be determined, was directly monitored amperometrically. For the second substrate we used not only 1 mM methyl viologen but also 1 mM benzyl viologen because its reduction potential at pH 7 (E_m,7) is less negative than that of the H₂/H⁺ couple and because in early work unusual behavior of methyl viologen was reported for this reaction (7) (see below). No activation was required for the enzyme kept anaerobically. Shortly (<5 s) after enzyme injection into the Clark cell, a steady rate of H₂ disappearance was observed, and this rate was considered the "initial" velocity value on the abscissa in Fig. 2. The data were fit well by simple Michaelis-Menten kinetics with a V_max of ca. 900 U/mg and a K_m for H₂ of 18 µM for the reaction with benzyl viologen as the electron acceptor and with a V_max of ca. 942 U/mg and a K_m for H₂ of 20 µM for the reaction with methyl viologen as the electron acceptor. The K_m values are the same order of magnitude as the K_0.5 for H₂ of 10^–5 M previously estimated in a progress curve analysis with methyl viologen as the acceptor (19).

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FIG. 1. P. furiosus soluble hydrogenase I has a relatively low Km for molecular hydrogen. (Upper panel) Michaelis-Menten plot of the H2 concentration for the reaction with benzyl viologen at 80°C and pH 8.0 (Km, 17.5 ± 1.5 µM; Vmax, 891 ± 21 U/mg). (Lower panel) Reaction with methyl viologen (Km, 20.1 ± 2.8 µM; Vmax, 942 ± 43 U/mg).

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FIG. 2. P. furiosus hydrogenase I has a regular Km value for viologen acceptors. (Upper panel) Michaelis-Menten plot for the reaction with oxidized benzyl viologen at 80°C and pH 8.0 (Km, 190 ± 46 µM; Vmax, 778 ± 62 U/mg). (Lower panel) Reaction with oxidized methyl viologen (Km, 126 ± 15 µM; Vmax, 1,361 ± 68 U/mg).

As an independent check, the concentration of the second substrate, oxidized viologen, was varied while the concentration of the first substrate, H₂, was kept constant at a saturating concentration. Previously, Ma et al. reported a 25-fold difference in the two V_max values for the reaction of H₂ with methyl viologen, and the higher value was the value obtained in the experiment in which the viologen concentration was varied and the H₂ concentration was constant (11, 19). Ma et al. report an extremely high K_m for oxidized methyl viologen (5 mM), which partially explains the large difference in the two V_max values. This high value would also imply that the standard assay of H₂ oxidation activity with 1 mM methyl viologen is a rather suboptimal assay (namely, one-sixth of V_max). Our results did not reproduce these previous reports. The solubility of hydrogen in water at 80°C under 1 atm of H₂ is 745 µM (4), which is more than 1 order of magnitude greater than the K_m values reported here for H₂.

The variable substrate (viologen) was detected directly by its optical absorption, and the resulting initial velocities again fit simple Michaelis-Menten kinetics (Fig. 2) for a V_max of ca. 800 U/mg and a K_m for oxidized viologen of 0.19 mM in the reaction with benzyl viologen and for a V_max of ca. 1,400 U/mg and a K_m for oxidized viologen of 0.13 mM in the reaction with methyl viologen. The K_m values imply that the H₂ variation experiments were done with a viologen concentration (1 mM) well above the viologen K_m and, therefore, that the two experiments (variation of H₂ and variation of viologen) should have provided similar values for V_max, which was indeed approximately the case.

Originally, the hydrogen oxidation activity of the soluble hydrogenase at 80°C was reported to be approximately fourfold lower than its hydrogen production activity (7). However, we later found that the production activity at 80°C was approximately the same as the consumption activity at 60°C, suggesting that the ratio of consumption activity to production activity at the same temperature is significantly greater than unity (16). Here we determined both of these activities at 80°C under standard conditions, and the hydrogen consumption activity was approximately eight times higher than the hydrogen production activity.

Complete insensitivity to CO at a concentration up to 320 µM both for H₂ uptake activity and for H₂ production activity has been reported for P. furiosus soluble hydrogenase (1, 2). In contrast, we found that in fact, the production activity at 80°C with reduced methyl viologen as the electron donor exhibited significant sensitivity to CO, with ca. 82% inhibition at 320 µM CO and a maximum level of inhibition of >97% in CO-saturated buffer (i.e., 691 µM CO), as shown in Fig. 3.

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FIG. 3. P. furiosus hydrogenase I is inhibited by carbon monoxide. The inhibition of H2 production by CO was measured at 80°C and pH 8.0. The solid line is a fit to the generic function for reversible inhibition of an enzyme obeying simple Michaelis-Menten kinetics at a constant initial substrate concentration: v = vmax/(1 + [CO]), and = 5,000 is a fitted lump parameter.

Finally, NADPH was proposed by Ma et al. to be the physiological electron donor for the soluble hydrogenase in H₂ production (12), but this proposal was questioned by Silva et al., who proposed that NADP⁺ is the electron acceptor for the enzyme in H₂ consumption (17). In the latter case one would expect the K_m for NADP⁺ to be in the micromolar range, and a value of 40 µM was indeed reported by Ma et al. (11, 12) We repeated this experiment and obtained a very similar K_m for NADP⁺, 37 µM (Fig. 4).

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FIG. 4. Reduction of NADP+ by H2 catalyzed by P. furiosus hydrogenase I. The Michaelis-Menten plot at 80°C and pH 8.0 gives a Km for NADP+ of 37 ± 2 µM and a Vmax of 34 ± 5 U/mg.

In conclusion, for the soluble, four-subunit hydrogenase I of P. furiosus we obtained a K_m for H₂ of ca. 20 µM, which is within the range of values reported for other NiFe-hydrogenases and, in particular, is similar to the value reported for the soluble, NAD⁺-reducing, multisubunit hydrogenase from Ralstonia eutropha, 37 µM (15). Also, the K_m values for the oxidized viologens (0.1 to 0.2 mM) are not at all unusual. Furthermore, in our hands the hydrogen consumption activity was 1 order of magnitude greater than the hydrogen production activity. Finally, during hydrogen production the enzyme exhibits rather significant sensitivity to CO inhibition. Taken together, these observations appear to show that the soluble P. furiosus enzyme does not function as a novel type of production enzyme (7, 11, 12), and they support the proposal that this enzyme is kinetically similar to the NiFe-hydrogenase congeners in other microorganisms. The present results corroborate the previous proposal that the soluble enzyme hydrogenase I is likely to function in the regeneration of NADPH at the expense of H₂ produced by the membrane-bound hydrogenase in proton respiration (16).

 
This research was supported by the European Commission through research project PYRED QLTR-2000-01676 and by the Dutch National Research School Collaboration-Catalysis as research project NRSCC-2004-10069.

 
* Corresponding author. Mailing address: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31 15 2785051. Fax: 31 15 2782355. E-mail: w.r.hagen{at}tudelft.nl

Published ahead of print on 21 December 2007.

Present address: Department of Agriculture and Food Sciences, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands.

Present address: Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal.

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Journal of Bacteriology, March 2008, p. 1584-1587, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01562-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by van Haaster, D. J. Articles by Hagen, W. R. Search for Related Content PubMed PubMed Citation Articles by van Haaster, D. J. Articles by Hagen, W. R.