The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

Abstract

Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1...

Ultrafast 2D-IR spectroscopy of [NiFe] hydrogenase from E. coli reveals the role of the protein scaffold in controlling the active site environment

“…27 When added to the biochemical characterization data reported above, this discrepancy adds further weight to our assignment of the as-isolated Ec Hyd-1 spectrum to different states with coincident frequencies to one of the substates referred to as Ni-R. 27 The frequency coincidence extends to the ν CN modes, which for both the Ni-R II and Ni-R III states were quoted as 2050 and 2067 cm −1 , 27 while a recent study in the crystalline phase identified four bands between 2049 and 2071 cm −1 , though without direct assignment to the sub-states. 91 Thus the fact that we identify a similar set of frequencies as being attributable to a particular state is consistent with the Ni r -S I/II and Ni-R II and Ni-R III states being spectroscopically very similar. There are in fact numerous reports of NiFe hydrogenases in distinct states exhibiting very similar IR signatures, for instance: Ni r -B and Ni u -A in Allochromatium vinosum MBH, 29 Desulfovibrio vulgaris Miyazaki F, 92 and Desulfovibrio gigas MBH ; 93 and Ni a -S and Ni r -S in Ralstonia eutropha MBH 94 and RH, 95 and Ni a -SR III and Ni a -SR II of Ralstonia eutropha SH.…”

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

“…2,10,18 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 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.…”

“…Although the way these species have been classified/named is not always consistent, three main species (i.e., Ni a -L1, Ni a -L2 and Ni a -L3) have been described and insights from experimental and theoretical works point toward the protonation of one of the Ni-bound terminal cysteines in some of these states. 7,16 25 This discrepancy suggests that different proton acceptors might coexist in [NiFe]-hydrogenases, with some reports speculating that such diversity might be related to the O 2tolerance/sensitivity of hydrogenases. 26 Herein, we employed cryogenic IR and EPR to resolve the longstanding debate about the structure of the Ni a -L1 and Ni a -L2 sub-forms, using CnRH as model hydrogenase.…”

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

“…Changes in the protonation state of a Ni-bound Cys (corresponding to Cys479 in Cn RH Figure S1) of the O 2 -sensitive Dv MF [NiFe]-hydrogenase have been shown by cryogenic IR investigations of the photoinduced transition from Ni a -C to Ni a -L2, an ultrahigh-resolution structure of the enzyme in the Ni a -SR states (78–85% Ni a -SR and 9–18% Ni a -SR′), and a combined DFT/NRVS study of the Ni a -SR species. , Conversely, IR spectroscopic experiments performed on the O 2 -tolerant Ec Hyd1 revealed no change in the protonation state of the corresponding cysteine (Cys576 in Ec Hyd1, Figure S1) in any of the Ni a -S, Ni a -C, Ni a -L2/3, or Ni a -SR′/″ states . This discrepancy suggests that different proton acceptors might coexist in [NiFe]-hydrogenases, with some reports speculating that such diversity might be related to the O 2 -tolerance/sensitivity of hydrogenases …”