IR spectroelectrochemical study of the binding of carbon monoxide to the active site of Desulfovibrio fructosovorans Ni-Fe hydrogenase

Abstract

The binding of carbon monoxide, a competitive inhibitor of many hydrogenases, to the active site of Desulfovibrio fructosovorans hydrogenase has been studied by infrared spectroscopy in a spectroelectrochemical cell. Direct evidence has been obtained of which redox states of the enzyme can bind extrinsic CO. Redox states A, B and SU do not bind extrinsic CO; only after reductive activation of the hydrogenase can CO bind to the active site. Two states with bound extrinsic CO can be distinguished by FTIR. These two states are in redox equilibrium and are most probably due to different oxidation states of the proximal 4Fe-4S cluster. Vibrational frequencies and theoretical quantum mechanics studies (DFT) of this process preclude the possibility of strong bonding of extrinsic CO to the Fe or Ni atoms of the active site. We propose that CO inhibition is caused by weak interaction of the extrinsic ligand with the Ni atom, blocking electron and proton transfer at the active site. A calculated structure with a weakly bound extrinsic CO at Ni has relative CO frequencies in excellent agreement with the experimental ones.

A model for the CO-inhibited form of [NiFe] hydrogenase: synthesis of (CO) 3 Fe( μ -S t Bu) 3 Ni{SC 6 H 3 -2,6-(mesityl) 2 } and reversible CO addition at the Ni site

“…Significantly, the Ni-CO stretching frequency for 3 (2055cm −1 ) is comparable to those observed for the CO-inhibited forms of [NiFe] hydrogenases from C. vinosum and Desulfovibrio fructosovorans, which exhibit the nickel-bound CO band in the 2055-2060cm −1 range (44,48). This similarity suggests that the electronic properties of the nickel centers in 3 and the CO-inhibited forms of the [NiFe] hydrogenases are similar.…”

Interaction of the active site of the Ni–Fe–Se hydrogenase from Desulfovibrio vulgaris Hildenborough with carbon monoxide and oxygen inhibitors

“…It has been shown previously by FTIR [25][26][27] and X-ray diffraction [20,21] vulgaris Ni-Fe-Se hydrogenase and their relative band intensities are very similar to those measured for CO-inhibited Ni-Fe hydrogenases [25][26][27]. Moreover, the fact that the intrinsic CO ligands are not vibrationally coupled with the extrinsic ligand clearly suggests that the later is coordinated to the Ni atom as in Ni-Fe hydrogenases [21].…”

“…It has been shown by different techniques, such as X-ray diffraction [1,[20][21][22], EPR [23], ENDOR [24] and FTIR [25][26][27] …”

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

“…The active site in [NiFe] hydrogenases has been characterized as a NiFe(CN) 2 (CO) center by x-ray crystallography (22,23,28,30) and by Fourier transform infrared spectroscopic (FTIR) 2 studies (4,21,(31)(32)(33), showing that the iron atom carries three inorganic diatomic ligands, two cyanides and one carbon monoxide. Nickel is coordinated by the sulfur atoms of four cysteines.…”