The exchange activities of [Fe] hydrogenase (iron-sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

Abstract

[Fe] hydrogenase (iron-sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min(-1) mg(-1)). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors.

Fig. 1

Reaction catalyzed by [Fe] hydrogenase. N⁵,N¹⁰-Methenyltetrahydromethanopterin (methenyl-H₄MPT⁺) reduction with H₂ to N⁵,N¹⁰-methylenetetrahydromethanopterin (methylene-H₄MPT) and a proton, whereby a hydride is stereospecifically transferred from H₂ into the pro-R side of methylene-H₄MPT [24]

Fig. 2

Detection limit of the method employed for the determination of the T₂/H₂O exchange activity of [Fe] hydrogenase. The assays were performed in 3.5-mL vials closed with a rubber stopper. The vials contained 1 mL standard assay mixture: 120 mM potassium phosphate pH 6.0, 1 mM EDTA and either no enzyme (open squares) or 0.5 mg (circles), 1.0 mg (triangles) or 10 mg (filled squares) purified [Fe] hydrogenase from Methanothermobacter marburgensis. The 2.5 mL gas phase consisted of 24% tritium-labeled H₂ (2.4 kBq μmol⁻¹) and 76% N₂ at 1.2 × 10⁵ Pa. At the times indicated, 0.1 mL liquid samples were withdrawn and analyzed for tritium radioactivity. From the specific radioactivity of tritium-labeled H₂ and the radioactivity above the background incorporated into water per 60 min, the exchange activity of the [Fe] hydrogenase in the 1 mL assay is calculated to be 0.35 mU (circles), 0.7 mU (triangles) and 8 mU (filled squares). The results show that the lower detection limit is near 0.1 mU. One unit is equivalent to 1 μmol H₂ exchanged into water per minute

Fig. 3

T₂/H₂O exchange activity of [Fe] hydrogenase in the presence and absence of methenyl-H₄MPT⁺. a Time dependence at different protein concentrations; b protein dependence. The assay conditions were essentially the same as described in the legend to Fig. 2. Instead of [Fe] hydrogenase from M. marburgensis, the assays contained the indicated microgram amounts of reconstituted [Fe] hydrogenase from Methanocaldococcus jannaschii (jHmd). Where indicated, the assays contained 5 μM methenyl-H₄MPT⁺. One unit is equivalent to 1 μmol H₂ exchanged into water per minute

Fig. 4

Dependence of the T₂/H₂O exchange activity of [Fe] hydrogenase on the methenyl-H₄MPT⁺ concentration at two different H₂ concentrations. The 1 mL assay contained 120 mM potassium phosphate pH 6.0, 1 mM EDTA, 2.0 μg reconstituted [Fe] hydrogenase from M. jannaschii and methenyl-H₄MPT⁺ at the concentrations indicated. The gas phase was either 24% tritium-labeled H₂/76% N₂ or 14% tritium-labeled H₂/78% N₂. The specific radioactivity of the tritium-labeled H₂ was 3.3 kBq μmol⁻¹. One unit is equivalent to 1 μmol H₂ exchanged into water per minute

Fig. 5

Mechanism that can explain the parallel formation of HD and H₂ from D₂ and H₂O as catalyzed by [Fe] hydrogenase in the presence of methenyl-H₄MPT⁺ (Fig. 6a). The carbocationic C(14a) of methenyl-H₄MPT⁺ bound to the enzyme is shown as C⁺. The carbocation is assumed to resemble a second transition metal center as do carbenes [49]. The catalytic cycle starts with the formation of the (η²-D₂)Fe complex, which is in electronic equilibrium with the cationic (μ-D)₂ complex. This is followed by an exchange of the (μ-D)₂ complex with protons of bulk water and either by a rotation of HD in the (η²-HD)Fe complex followed by a second exchange or by the dissociation of the complex with the release of HD. The parallel formation of HD and H₂ at equal rates (Fig. 6a) can be explained assuming that k_off = k_exchange and that all other steps in the catalytic cycle are not rate limiting

Fig. 6

Kinetics of H₂ and HD formation from D₂ and H₂O catalyzed a by [Fe] hydrogenase from M. marburgensis and b by the H₂-signaling [NiFe] hydrogenase from Rhodobacter capsulatus. a The 7 mL assay mixtures at 40 °C contained 120 mM potassium phosphate pH 6.0, 7 μM methenyl-H₄MPT⁺ and 150 μM D₂ (20% saturation). The reactions were started by the addition of 0.13 U purified [Fe] hydrogenase from M. marburgensis. Dihydrogen isotopes were determined on line by mass spectrometry. b The 1.5 mL assay mixtures at 30 °C contained 50 mM citrate–phosphate pH 7.0 and cells of R. capsulatus mutants containing only the H₂-signaling hydrogenase. The suspension was gassed with 100% D₂ before the vessel was closed and the formation of HD and that of H₂ were monitored by mass spectrometry. (a The data were taken from [27] with permission; b the data were taken from [67] with permission)