Stepwise assembly of the active site of [NiFe]-hydrogenase

Abstract

NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2 eand 2 H + under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CNmolecules. Here, we investigated the sequential assembly of the [NiFe]-cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox-inactive, and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, Xray absorption, and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

“…Ni a -C has been shown to photoconvert into Ni a -L species in several [NiFe]-hydrogenases, , but the latter intermediate was detected almost exclusively at cryogenic temperatures due to the rapid Ni a -L to Ni a -C back-conversion. ,, However, in a few studies, CO and CN absorptions associated to Ni a -L intermediates have been observed also at ambient temperature. Examples are (i) time-resolved and steady state IR spectroscopic studies on Pf SH1 hydrogenase and its R355K variant, ,, (ii) spectroelectrochemical and pH-dependent studies on Ec Hyd1 protein solutions and single crystals, ,,, and (iii) IR studies on isolated C. necator hydrogenase large subunits treated with reducing agents. , …”

“…The Fe is additionally ligated by one CO and two CN – (strong-field) ligands, which stabilize a low-spin Fe II configuration (Figure ). , Infrared (IR) spectroscopy has played a pivotal role in characterizing the various active and inhibited states of hydrogenases, observing shifts in the vibrational bands of the active site CO and CN – ligands due to structural and redox changes . Recent achievements in this regard have been summarized elsewhere. − The O 2 -tolerant regulatory [NiFe]-hydrogenase (RH) from the ’Knallgas’ bacterium Cupriavidus necator ( Cn ) has been used as a model enzyme to elucidate the electronic and molecular structure of certain catalytic intermediates.…”

“…The Fe is additionally ligated by one CO and two CN − (strongfield) ligands, which stabilize a low-spin Fe II configuration (Figure 1). 4,5 Infrared (IR) spectroscopy has played a pivotal role in characterizing the various active and inhibited states of hydrogenases, observing shifts in the vibrational bands of the active site CO and CN − ligands due to structural and redox changes. 6 Recent achievements in this regard have been summarized elsewhere.…”

Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

“…Examples are i) time-resolved and steady state IR spectroscopic studies on PfSH1 hydrogenase and its R355K variant, 17,21,30 ii) spectroelectrochemical and pH-dependent studies on EcHyd1 protein solutions and single crystals, 20,25,26,31 and iii) IR studies on isolated C. necator hydrogenase large subunits treated with reducing agents. 5,28 Irradiation of RH samples enriched in the Ni a -C state at 90 K (pH 8.0) with an LED array (460 nm) results in the enrichment of a single species, Ni a -L1, characterized by a ν CO band at 1911 cm -1 and ν CN stretches at 2037 and 2056 cm -1 (black trace in Fig. 2a, Table S1).…”

“…1). 4,5 Infrared (IR) spectroscopy has played a pivotal role in characterizing the various active and inhibited states of hydrogenases, observing shifts in the vibrational bands of the active site CO and CN − ligands due to structural and redox changes. 6 Recent achievements in this regard have been summarized elsewhere.…”