A Vertebrate-type Ferredoxin Domain in the Na+-translocating NADH Dehydrogenase from Vibrio cholerae

Abstract

The Na؉ -translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mö ssbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys 70 , Cys 76 , Cys 79 , and Cys 111 . Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.Iron-sulfur proteins are present in all domains of living organisms where they exhibit diverse functions like electron transport, catalysis, and sensing in regulatory processes (1). In some NADH-oxidizing, respiratory complexes, Fe-S centers accept electrons from flavin cofactors in an overall exergonic reaction that results in the reduction of quinone. This electron transfer reaction drives the uphill transport of protons or Na ϩ across the inner membrane of mitochondria or bacteria. The NADH:quinone oxidoreductase (Na ϩ -NQR) 1 from the human pathogen Vibrio cholerae maintains an electrochemical Na ϩ gradient across the inner bacterial membrane, which strongly influences the production of virulence factors (2). The Na ϩ -NQR consists of six subunits, NqrA-F, and contains one Fe-S center, two covalently bound FMNs, one non-covalently bound FAD, one riboflavin, and ubiquinone-8 as prosthetic groups (3-7). The NqrF subunit of the Na ϩ -NQR complex is anchored to the inner membrane and displays a clearly defined domain structure. The N-terminal Fe-S domain harbors the [2Fe-2S] cluster, while the binding sites for the non-covalently bound FAD and NADH are located in the C-terminal domain of NqrF. The initial oxidation of NADH by the NqrF subunit results in the two-electron reduction of the FAD followed by one-electron transfer steps to the [2Fe-2S] cluster in the Fe-S domain (7). Here we study the electronic properties of the [2Fe-2S] cluster in the isolated Fe-S domain of NqrF. A comparison of its amino acid sequence with sequences of [2Fe-2S] ferredoxins from vertebrates and plants reveals that the Fe-S domain is related to ferredoxins of the vertebrate-type family. Vertebrate-type ferredoxins are soluble redox carriers that accept electrons from specific NADH:ferredoxin reductases and deliver them to enzymatic systems catalyzing the hydroxylation of various compounds like steroids or camphor (8). The Fe-S domain exhibits highest sequence similarity to vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters (ISC-type ...

Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production inVibrio cholerae

“…The electron transfer pathway in the Na ϩ -NQR starts by a hydride transfer from the substrate NADH to the non-covalently bound FAD on the flavin domain of the NqrF subunit (41), followed by one-electron transfer to the vertebrate-type [2Fe-2S] cluster in the N-terminal domain of NqrF (25). How electron transport from this FeS center to other flavin and ubiquinone cofactors in the complex and to the substrate quinone proceeds and how this overall exergonic reaction is coupled to Na ϩ transport are still enigmatic.…”

Oxidant-induced formation of a neutral flavosemiquinone in the Na+-translocating NADH:Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae

“…The presence of an additional riboflavin cofactor in the Na + -NQR is discussed controversially [8,9]. The electron transfer pathway in the Na + -NQR starts with a hydride transfer from the substrate NADH to the non-covalently bound FAD on the flavin domain of the NqrF subunit [10], followed by one-electron transfers to the vertebrate-type [2Fe-2S] cluster in the Nterminal domain of NqrF [11]. How electron transport from this FeS centre to other flavin and ubiquinone cofactors in the complex and to the substrate quinone proceeds and how this reaction is coupled to Na + transport is still enigmatic.…”

Electrostatics and water occlusion regulate covalently‐bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR

“…12 NQR is composed of six subunits (A-F) and five confirmed redox cofactors (Figure 1A,B): flavin adenine dinucleotide (FAD), a 2Fe-2S center, two covalently-bound flavin mononucleotide (FMN) molecules, and a riboflavin molecule. [8][9][10][11][13][14][15][16][17][18] Crystallographic data indicate that a sixth cofactor, a non-heme iron center, could also participate in electron transfer. 19,20 However, its participation in the electron transfer pathway has not been experimentally corroborated.…”