[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

Abstract

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

Figure 1

Alignment of amino acid sequences of various [NiFe] hydrogenase small subunits around the three cysteine ligands to the [3Fe-4S] center. The sequences were taken from refs. (D. gigas [NiFe] hydrogenase, D. g.), 18 (D. fructosovorans, D. f.), 21 (D. vulgaris Miyazaki F, D. v.), 22 (Rhodobacter capsulatus, R. c.), 23 (Bradirhizobium japonicum, B. j.), and 8 (D. baculatum, Dm. b.). The cysteine cluster ligands and the conserved proline residue corresponding to position 239 in D. gigas enzyme are shown in bold.

Figure 2

EPR spectra given by the hydrogenases from D. fructosovorans in the oxidized state. (a and c) Wild-type enzyme. (b and d) P238C mutant. Conditions: temperature, 12 K; microwave frequency, 9.422 GHz; microwave power, 0.04 mW; modulation amplitude, (a and b) 0.1 mT, (c and d) 1 mT.

Figure 3

EPR spectra given by reduced hydrogenases from D. fructosovorans. (a and c) Wild-type enzyme. (b and d) P238C mutant. Conditions: temperature, 6 K; microwave frequency, 9.420 GHz; microwave power, (a and b) 0.1 mW, (c and d) 1 mW; modulation amplitude, 1 mT.

Figure 4

Three-fold averaged electron density maps (45) calculated with Fo-Fc coefficients in the 20–3 Å resolution range and contoured at the 3.2 σ level (a and b) or with 2Fo-Fc coefficients and contoured at the 1.1 σ level (c). (a) Fo-Fc map calculated with the native model in which Pro-238 has been substituted by a glycine. The map confirms the presence of a mutation close to the [3Fe-4S] cluster, as there is a significant electron density peak, which is not explained by the native model. (b) Fo-Fc map calculated with the native model in which Pro-238 has been substituted by a cysteine. (c) 2Fo-Fc map calculated with the final model of the mutant including both the Cys-238 and the third [4Fe-4S] cluster.