Spectroscopic Insights into the Oxygen-tolerant Membrane-associated [NiFe] Hydrogenase of Ralstonia eutropha H16

Abstract

Hydrogenases are metalloenzymes that catalyze the reversible cleavage of H 2 into protons and electrons and play a pivotal role in the energy metabolism of many microorganisms (1). They are grouped into three phylogenetically distinct classes as follows: the di-iron [FeFe], nickel-iron [NiFe], and iron-sulfur cluster-free [Fe] hydrogenases (2-6). The basic module of [NiFe] hydrogenases consists of two subunits, a large subunit that contains the [NiFe] active site and a small subunit that accommodates one to three electron-transferring iron-sulfur

[NiFe] and [FeS] Cofactors in the Membrane-Bound Hydrogenase of Ralstonia eutropha Investigated by X-ray Absorption Spectroscopy: Insights into O2-Tolerant H2 Cleavage

“…Three different MBH protein preparations were used for this study. The most active MBH (mean activity of 14013 U/mg) was isolated from R. eutropha strain HF649, which was grown on fructose-glycerol mineral medium under oxygen-limited conditions (43)(44)54). The membrane fraction was prepared under an argon atmosphere and oxidized by the addition of 50 mM K 3 [Fe(CN) 6 ] prior to aerobic solubilisation with Triton X-114.…”

“…Spectroscopic studies on the MBH using EPR and FTIR techniques uncovered several features differing from those of standard enzymes (43)(44)(45)(46). The so-called Ni-A state, corresponding to oxidized, inactive unready enzyme, was not detectable in wild-type MBH.…”

“…According to the crystal structures of several [NiFe]-hydrogenases, generally four conserved cysteines in the large subunit are involved in coordinating the active site Ni and Fe atoms (27,41). A further common feature of [NiFe] H 2 ases is the ligation of the Fe by two cyanides (CN -) and one CO as observed by FTIR spectroscopy (36,(42)(43). Apart from these similarities, biochemical, electrochemical, and spectroscopic results (26,36,(43)(44)(45) suggest that O 2 tolerance of the MBH is related to the structural arrangement of its metal cofactors, rather than to the restricted access of O 2 and CO to the [NiFe] site due to a narrow gas-channel as proposed for H 2 -sensing H 2 ases (12,26,46).…”

“…Remarkably, in addition to the absence of Ni-A, a complex EPR spectrum indicates modification of the FeS cluster in the proximal position to the [NiFe] site in the MBH (43)(44)(45)(46). The structural basis of these modifications needs to be elucidated.…”

“…A further common feature of [NiFe] H 2 ases is the ligation of the Fe by two cyanides (CN -) and one CO as observed by FTIR spectroscopy (36,(42)(43). Apart from these similarities, biochemical, electrochemical, and spectroscopic results (26,36,(43)(44)(45) suggest that O 2 tolerance of the MBH is related to the structural arrangement of its metal cofactors, rather than to the restricted access of O 2 and CO to the [NiFe] site due to a narrow gas-channel as proposed for H 2 -sensing H 2 ases (12,26,46). Notably, compared to standard H 2 ases, biosynthesis of active MBH requires a significantly larger set of maturation proteins (47)(48)(49).…”