The Auxiliary Protein HypX Provides Oxygen Tolerance to the Soluble [NiFe]-Hydrogenase of Ralstonia eutropha H16 by Way of a Cyanide Ligand to Nickel

Abstract

The hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the [NiFe]-hydrogenases in the bacterium. A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD ؉ -reducing hydrogenase. The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypXnegative strain were compared with those from an enzyme purified from a HypX-positive strain. In activity assays under anaerobic conditions both enzyme preparations behaved the same. Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H 2 oxidation with NAD ؉ or benzyl viologen as the electron acceptor. Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme. The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide. The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions. 2 from the ␤-proteobacterium Ralstonia eutropha H16. This is a heterotetrameric enzyme with subunits HoxF (67 kDa), HoxH (55 kDa), HoxU (26 kDa), and HoxY (23 kDa) (9, 10). The enzyme comprises two functionally different heterodimeric complexes, which have been separated and characterized (9, 11). The HoxFU dimer constitutes an enzyme module, termed NADH dehydrogenase or diaphorase, involved in the reduction of NAD Fig. 1A. The bridging oxygen ligand is removed upon reduction, whereby these enzymes become activated (17,24).The SH of R. eutropha belongs to a subclass of [NiFe]-hydrogenases where the polypeptide of the small hydrogenase subunit (HoxY) ends shortly after the position of the fourth Cys residue coordinating the proximal cluster. The large hydrogenase subunit (HoxH) of the SH contains the four Cys residues, conserved in all [NiFe]-hydrogenases, for the binding of the

Oxygen Tolerance of the H2-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active Site

“…First, an additional CN Ϫ ligand bound to the nickel of the [NiFe] site contributes to the oxygen tolerance of the enzyme. Mutant proteins devoid of the nickel-bound CN Ϫ ligand turned out to be oxygen-sensitive (13,41). Second, x-ray absorption spectroscopy revealed that in contrast to standard [NiFe] hydrogenases the active site of the SH is coordinated by more oxygen ligands and by less sulfur ligands.…”

“…In the case of SH, two additional cyanide ligands, attached to the [NiFe] site, one bound to the iron and one bound to the nickel, have been identified by Fourier-transformed infrared spectroscopy (12). The lack of the nickel-bound CN Ϫ in a mutant protein correlated with the occurrence of oxygen sensitivity of the SH suggesting that the modification of the active site accounts for the oxygen tolerance of this specific enzyme (13). On the other hand, based on spectroscopic and chemical data, the RH of R. eutropha shows a standard [NiFe] site so far as the number of CO and CN Ϫ ligands is concerned (14), raising the question as to what kind of mechanism may account for the oxygen tolerance of this hydrogenase.…”

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H 2 -Oxidizing Bacteria

“…1) is essential for group 5 [NiFe]-hydrogenase activity or not remains to be established, especially for hypX and ORFs 1 to 4, whose physiological function is unknown. The hypX-like gene encodes a putative protein, HypX, that has been shown to confer oxygen tolerance on the soluble [NiFe]-hydrogenase in R. eutropha H16 through the attachment of an additional cyanide ligand to the active site (6) and to participate in the maturation of the large subunit of the H 2 uptake hydrogenase in Rhizobium leguminosarum (45). The hypX gene was detected only in the hydrogenase gene clusters of Streptomyces representatives ( Table 1 HypA and HypB are involved in nickel storage and insertion (7,34,57).…”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…188 The oxidative inactivation of NiFe hydrogenases, a process first characterized in the mid-1980s, 415 has recently become a very hot topic because this reaction is the main obstacle to using these enzymes in biofuel cells 416,417 or in photosynthetic hydrogen production processes. Hence, much effort is currently devoted to identifying enzymes that can function in air 416 or to understanding the molecular bases of the oxygen sensitivity [418][419][420] in order to use genetic engineering techniques to design oxygen-resistant enzymes. 421 The prototypical NiFe hydrogenases from Allochromatium Vinosum or DesulfoVibrio species exist in a mixture of oxidized, inactive forms when they are purified under aerobic conditions, and activation requires reduction.…”