Selective release and function of one of the two FMN groups in the cytoplasmic NAD⁺‐reducing [NiFe]‐hydrogenase from Ralstonia eutropha

Abstract

The soluble, cytoplasmic NAD + -reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H 2 -NAD + reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity -the reduction of NAD + by H 2 . This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. [14,15]. The SH of R. eutropha belongs to a subclass of [NiFe]-hydrogenases where the polypeptide of the small hydrogenase subunit ends shortly after the position of the fourth Cys residue co-ordinating the proximal cluster [4]. The large HoxH subunit in the SH contains all conserved amino acid residues for binding of the Ni-Fe site [23,24]. Hence, the amino acid sequence suggests that the hydrogenase module in this enzyme only contains the Ni-Fe site and the proximal cluster as prosthetic groups. Fourier-transform infrared (FTIR) studies on the SH indicated that the Ni-Fe site contains two more CN ligands than the active site in standard hydrogenases, and is a (RS) 2 (CN)Ni(l-RS) 2 Fe(CN) 3 (CO) centre [25]. In contrast to standard hydrogenases, the SH is not sensitive towards oxygen and carbon monoxide and shows no redox changes of the Ni-Fe site. The Fe-S clusters in the HoxFUY subunits and the flavin in the HoxF subunit are all considered to be functional in the intramolecular electron transfer during the H 2 -NAD + reaction. It was shown recently that the protein content of SH preparations is considerably overestimated by the routine colourimetric protein-determination methods. This led to the finding that the SH contains two FMN groups and one NADH-reducible [2Fe-2S] cluster [26]. In the present paper we have investigated the possible role of the two FMN Correspondence to S. P. J. Albracht,

The Soluble NAD+-Reducing [NiFe]-Hydrogenase fromRalstonia eutrophaH16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

“…Infrared spectroscopic studies, chemical analyses, and recent X-ray absorption studies suggest that the inactive SH has a (CysSO) 2 (CN)(OOH)Ni(-CysS) 2 Fe(CN) 3 (CO) site where both metal ions are six coordinated (14,25,72,73). It has been suggested that the oxygen ligand to nickel is removed upon activation of the SH (25,44,72). For reasons reported elsewhere (14), we favor a peroxide group as ligand to nickel.…”

“…The HoxY subunit of the SH is a truncated version of the small subunit of standard [NiFe]-hydrogenases and presumably accommodates only the proximal [4Fe-4S] cluster. Recently, a second FMN (called FMN-a) was identified in the SH (72). It was postulated that the hydride formed upon H 2 cleavage at the Ni-Fe site is transferred to FMN-a, from where the electrons proceed via the Fe-S clusters to FMN-b and finally to NAD ϩ .…”

“…The SH consists of four heterologous subunits forming two functional modules: a hydrogenase dimer encoded by the hoxH and hoxY genes and an NADH-dehydrogenase dimer encoded by hoxF and hoxU. The latter moiety provides the NAD ϩ -binding site and harbors several Fe-S clusters in addition to one flavin mononucleotide (FMN) cofactor (called FMN-b [41,69,72]). The HoxH subunit of the SH contains six conserved motifs, which are typical of [NiFe] hydrogenases.…”

Mammalian NADH:ubiquinone oxidoreductase (Complex I) and nicotinamide nucleotide transhydrogenase (Nnt) together regulate the mitochondrial production of H2O2—Implications for their role in disease, especially cancer

“…This major achievement disclosed the long-awaited information on the relative positions of the subunits and prosthetic groups involved in electron transfer. In contrast to the native bovine enzyme, which contains 2 FMN groups (Albracht et al 2003;Van der Linden et al 2004), the crystallized T. Rot Fig. 1 The prosthetic groups in bovine Complex I, together with the proposed reaction sites of NADH, NADPH, rotenone and ubiquinone, and the observed pH dependencies of the NAD(P)H→O 2 reactions of submitochondrial particles.…”

“…30 min, and an incubation of the SMP with 1 mM NADH for 60 min, may lead to partial (reversible) release of FMN from Complex I as demonstrated by Vinogradov and coworkers (Gostimskaya et al 2007). Reduction of the R. eutropha soluble NAD + -reducing [NiFe]-hydrogenase by NADH at pH 8 results in the specific rapid (reversible) release of FMN-a, whereas FMN-b remains firmly bound (Van der Linden et al 2004).…”