Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii

Abstract

[FeFe] hydrogenase from Clostridium beijerinkii (CbHydA1) is an unusual hydrogenase in that it can withstand prolonged exposure to O2 by reversibly converting into an O2-protected, inactive state (Hinact). It has been indicated in the past that an atypical conformation of the “SC367CP” loop near the [2Fe]H portion of the six-iron active site (H-cluster) allows the Cys367 residue to adopt an “off-H+-pathway” orientation, promoting a facile transition of the cofactor to Hinact. Here, we investigated the electronic structure of the H-cluster in the oxidized state (Hox) that directly converts to Hinact under oxidizing conditions and the related CO-inhibited state (Hox-CO). We demonstrate that both states exhibit two distinct forms in electron paramagnetic resonance (EPR) spectroscopy. The ratio between the two forms is pH-dependent but also sensitive to the buffer choice. Our IR and EPR analyses illustrate that the spectral heterogeneity is due to a perturbation of the coordination environment of the H-cluster’s [4Fe4S]H subcluster without affecting the [2Fe]H subcluster. Overall, we conclude that the observation of two spectral components per state is evidence of heterogeneity of the environment of the H-cluster likely associated with conformational mobility of the SCCP loop. Such flexibility may allow Cys367 to switch rapidly between off- and on-H+-pathway rotamers. Consequently, we believe such structural mobility may be the key to maintaining high enzymatic activity while allowing a facile transition to the O2-protected state.

Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis: An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase

“…In the latter case, two distinct EPR signals were observed for the H ox state, but only a single set of FTIR bands, an observation attributed to structural flexibility around the [4Fe–4S] component of the H-cluster. 47 Assuming that State 2 does indeed correspond to a similar mixed valence I,II formal oxidation state of the [2Fe] H subsite, the shift from rhombic to axial EPR signal as well as the distinct differences observed in the FTIR spectra indicates a change in the geometry and/or electron configuration of the H-cluster as compared to the standard H ox state. 9 Moreover, State 2 accumulates under H 2 in at least three of the variants (E289A/D and E252V), while incubation under a CO atmosphere results in (slow) conversion to State 1 .…”

Catalytic Bias and Redox-Driven Inactivation of the Group B FeFe Hydrogenase CpIII

“…There has been much interest recently in the reaction of the H-cluster of prototypical ,, and atypical hydrogenases , from group A (Cr HydA1 and Cb, respectively) with extrinsic or intrinsic sulfide. The formation of the sulfide-bound oxidized inactive state is revealed by a particular FTIR signature called H inact and reversed upon reduction.…”

“…Reversible oxidative inactivation occurs withC. beijerinckii FeFe hydrogenase (Cb, also from group A, gray in Figure ) in the absence of chloride or exogenous sulfide, and it results from the formation of the H inact state upon binding to the distal Fe of the H-cluster of the sulfur of the cysteine that is equivalent to CpI C299 ,, (see Figure A). From the observation that CpIII is also oxidized to a state whose signature is very similar to that of the H inact state in the absence of sulfide (Table S3 in ref ), we hypothesize that the binding of the cysteine to Fe d occurs in CpIII, as it does in Cb.…”

Catalytic bias and redox-driven inactivation of ancestral FeFe hydrogenases from group B2

“…There has been much interest recently in the reaction of the H-cluster of standard 16,17,30 and non-standard hydrogenases 11,31 from group A (Cr and Cb, respectively) with extrinsic or intrinsic sulfide. The formation of the sulfide-bound oxidized inactive state is revealed by a particular FTIR signature called H inact and reversed upon reduction.…”

“…Reversible oxidative inactivation occurs with Clostridium beijerinckii FeFe hydrogenase (Cb, also from group A, gray in fig 2) in the absence of chloride or exogenous sulfide, and it results from the formation of the Hinact state upon binding to the distal Fe of the H-cluster of the sulfur of the cysteine that is equivalent to CpI C299 11,31,32 (see figure 1A). The observation that CpIII is also oxidized to the Hinact state in the absence of sulfide (Table S3 in ref 5 ) strongly suggests that the binding of the cysteine to Fe d occurs in CpIII, as it does in Cb.…”