Structural Studies of the Carbon Monoxide Complex of [NiFe]hydrogenase fromDesulfovibriovulgarisMiyazaki F:  Suggestion for the Initial Activation Site for Dihydrogen

Abstract

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.

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

“…On the other hand, some discrepancies are found in the number of thiolates between metals (three for 3 and two for hydrogenase) and the diatomic ligands on iron (FeðCOÞ 3 for 3 and FeðCOÞðCNÞ 2 for hydrogenase). Furthermore, the Ni-Fe distance in 3 (3.17 Å) is substantially longer than those in the protein (2.61-2.62 Å) (42). Perhaps the oxidation states of Fe and/or Ni of 3 are different from those of the structurally characterized CO-inhibited form of [NiFe] hydrogenase.…”

“…According to a crystallographic analysis of the CO-inhibited form of the [NiFe] hydrogenase obtained from Desulfovibrio vulgaris Miyazaki F (D. v. Miyazaki F), a CO molecule is coordinated at the nickel center (42). It has been suggested that this nickel-bound CO can be liberated and the catalytic activity recovered, upon flushing with a stream of N 2 (43), or by white-light irradiation at 20 K (44 (45)(46)(47).…”

Electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels

“…2.71 Å for H 2 , calculated from gas viscosities) (20); consequently, CO must also have access, and the fact that it does not inhibit suggests an inability to form a metal-CO bond. According to structural and spectroscopic studies carried out on the standard [NiFe] hydrogenase from D. vulgaris (Miyazaki F), the exogenous (inhibitory) CO molecule binds to the Ni atom (21). It is thus important to know how typical is the active site of Re MBH.…”

Solar water-splitting into H2 and O2: design principles of photosystem II and hydrogenases

“…Indeed, comparative crystallography showed that CO and O 2 inhibited [NiFe] hydrogenases show additional diatomic electron density at the open fifth coordination site at Ni. 138 In the oxidized ''unready'' state of the enzyme, an additional elongated electron density was observed at the bridging ligand ''X'', suggesting it to be a hydroperoxide OOH À . 128 It is suggested that the peroxo bridging ligand causes the enzyme to be ''locked'' in the unready state which can be reactivated only very slowly.…”