Impact of Amino Acid Substitutions near the Catalytic Site on the Spectral Properties of an O₂‐Tolerant Membrane‐Bound [NiFe] Hydrogenase

Abstract

[NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain further insight into the mechanism(s) responsible for the remarkable O(2) tolerance, we employ FTIR and EPR spectroscopy to study mutant variants of the membrane-bound hydrogenase (MBH) of Re-carrying substitutions of a particular cysteine residue in the vicinity of the [NiFe] active site that is characteristic of O(2)-tolerant membrane-bound [NiFe] hydrogenases. We demonstrate that these MBH variants, despite minor changes in the electronic structure and in the interaction behavior with the embedding protein matrix, display all relevant catalytic and noncatalytic states of the wild-type enzyme, as long as they are still located in the cytoplasmic membrane. Notably, in the oxidized Ni(r)-B state and the fully reduced forms, the CO stretching frequency increases with increasing polarity of the respective amino acid residue at the specific position of the cysteine residue. We purified the MBH mutant protein with a cysteine-to-alanine exchange to apparent homogeneity as dimeric enzyme after detergent solubilization from the membrane. This purified version displays increased oxygen sensitivity, which is reflected by detection of the oxygen-inhibited Ni(u)-A state, an irreversible inactive redox state, and the light-induced Ni(a)-L state even at room temperature.

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

“…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).…”

“…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.…”

“…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.…”

Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

“…In addition, a considerable amount of spectral heterogeneity was detected. The maximum of the CO absorption was only slightly shifted (⌬ Ϸ ϩ1 cm Ϫ1 ) compared with Ni r -B, which can be explained by an overlap of absorptions characteristic for Ni u -A and Ni r -B species (11,46). Furthermore, prominent shoulders attributable to the EPR-silent inactive states Ni u -S and Ni ia -S (22) (Fig.…”

Insights into the structure of the active site of the O2-tolerant membrane bound [NiFe] hydrogenase of R. eutropha H16 by molecular modelling

“…10 A similar procedure has been employed in the past for determining the structure of a guanosine triphosphate (GTP) ligand bound to a Ras protein. 16 Calculated spectra were obtained from 25 snapshots of the MD trajectory.…”