A New Flavin Radical Signal in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Self Cite

103

Here we present new evidence that riboflavin is present as one of four flavins in Na ؉ -NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na ؉ -NQR.The Na ϩ

contrasting

confidence: 55%

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

Juárez

Nilges

Gillespie

et al. 2008

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103

“…An open question concerning the mechanism of Na ϩ -NQR is how one-electron transfer reactions initiated by the 2Fe-2S cluster on NqrF are coupled to the two-electron reduction of quinone to quinol (2,4,8,67). Both riboflavin and the covalently linked FMNs were proposed to exclusively act as one-electron converters in Na ϩ -NQR (4,(67)(68)(69). Our finding that both riboflavin and one covalently bound FMN are located in NqrB suggests that these cofactors are in electron-transfer distance and supports the view that NqrB catalyzes the ultimate reduction steps leading to formation of ubiquinol from ubiquinone.…”

Section: Discussionmentioning

confidence: 99%

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

Casutt

Huber

Brunisholz³

et al. 2010

The sodium ion-translocating NADH:quinone oxidoreductase (Na ؉ -NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na ؉ across the bacterial membrane. The Na ؉ -NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na ؉ -NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na ؉ -NQR is discussed.Pathogenic strains of the water-borne bacterium Vibrio cholerae cause the diarrheal disease cholera. By the help of a respiratory Na ϩ pump, V. cholerae maintains an electrochemical Na ϩ gradient across the inner membrane to drive central processes like flagellar rotation, nutrient uptake, and detoxification. The respiratory Na ϩ pump, also called Na2 is a membrane-bound enzyme complex composed of six subunits (NqrABCDEF) (1, 2). NqrA is a peripheral subunit, NqrC and NqrF are predominantly hydrophilic yet anchored to the membrane, whereas the hydrophobic NqrB, NqrD, and NqrE subunits each comprise several transmembrane helices. The Na ϩ -NQR contains at least five distinct redox centers participating in electron transfer from NADH to substrate quinone: noncovalently bound FAD, a 2Fe-2S cluster, covalently bound FMNs, and noncovalently bound riboflavin (3-5). In addition, ubiquinone-8 (Q 8 ) was co-purified with the Na ϩ -NQR (6). Binding sites for FAD, 2Fe-2S cluster, and substrate NADH are located on NqrF, which does not directly participate in Na ϩ -transport but represents the electron input module of the complex catalyzing the NADH dehydrogenase reaction (7,8). The covalently bound FMN residues are attached via phosphodiester bonds to Thr-236 in subunit NqrB and Thr-225 in subunit NqrC, respectively (9, 10). The subunit(s) responsible for riboflavin and quinone binding have not been identified yet. Riboflavin was detected in a subcomplex composed of NqrA, -B, -C, and -F but was not present in the isolated A and F subunits (3). Furthermore, there is evidence that NqrB participates in the quinone reduction step (11).By analyzing the flavin cofactors in defined subcomplexes of the Na ϩ -NQR from V. cholerae, we show that riboflavin is localized on subunit NqrB. Analysis of the molar size and subunit composition of the holo-complex reveals that the Na ϩ -NQR contains one copy of each subunit and a total ...

“…An EPR signal at g ϭ 2.00 (Fig. 7A), which may reflect flavo-semiquinone radicals (59,60), was intensified when BchX was exposed for 3-5 min to air after reduction with dithionite (data not shown), indicative of the presence of FMN.…”

Section: Suppressor Mutations Rescuing the Inability Of Sodb1 To Growmentioning

confidence: 99%

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Kim

Lee

et al. 2008

Chlorophyllide a reductase of Rhodobacter sphaeroides, which were reconstituted with the purified subunits of BchX, BchY, and BchZ, reduced ring B of chlorophyllide a using NADH under anaerobic conditions. Interestingly, suppressor mutations rescuing the inability of R. sphaeroides Fe-SOD mutant to grow in succinate-based minimal medium were predominantly mapped to BchZ subunit of chlorophyllide a reductase. The enzyme is labile in the presence of O 2 . However, it generates superoxide at low O 2 . The enzymes reconstituted with BchX, BchY, and the mutein subunit of BchZ from suppressor mutants showed less activity not only for chlorophyllide a reduction but also for superoxide generation compared with the enzyme reconstituted with the wild-type subunits. BchX, which contains FMN, and BchY are iron-sulfur proteins, whereas BchZ is a hemoprotein containing b-type heme. Neither chlorophyllide a reduction nor superoxide generation was observed with the enzyme reconstituted with the wild-type subunits of BchX and BchY, and the apo-subunit of BchZ that had been refolded without heme, in which FMN of BchX was fully reduced. Thus, superoxide is generated not from FMN of BchX but from heme of BchZ. Consistently, the heme of BchZ muteins was half-reduced in its redox state compared with that of wild-type BchZ.Rhodobacter sphaeroides, a facultative photosynthetic bacterium, contains two SODs 3 ; CuZn-SOD is detected only under the conditions where photosynthetic complexes are formed (1), whereas Fe-SOD is constitutively expressed, although its activity of the aerobically grown cell nearly doubled as compared with that of the anaerobically grown cell. The role of CuZn-SOD in protecting the photoheterotrophic cells from periplasmic superoxide upon exposure to O 2 was proposed (1). A cambialistic SOD using manganese or iron is the only SOD found in Rhodobacter capsulatus and is essential for cell viability (2, 3). Oxidative stress defense in R. sphaeroides and R. capsulatus is also mediated by catalases as well as by glutathione-glutaredoxin and thioredoxin systems that act as thiol-disulfide redox buffer to reduce the protein thiols that were oxidized (4, 5). Mutations in glutathione-glutaredoxin and thioredoxin systems lowered the formation of the photosynthetic complex (4, 5).The formation of photosynthetic complexes of R. sphaeroides is redox-dependent. Lowering oxygen tension induces the formation of intracytoplasmic membrane housing the photosynthetic complexes of R. sphaeroides. Light captured by B800 -850 and B875 LH complexes is transferred to reaction center complex, where the redox reactions are initiated to convert light energy into ATP and reducing power. The oxygenregulated expression of apoproteins of LH and reaction center complexes, which are encoded by puc, puf, and puh operons, was elucidated (for reviews see Refs. 6 and 7).The expression of several enzymes for Bchl a synthesis is also subject to anaerobic induction (8 -10). Protoporphyrin IX, which is synthesized from 5-aminolevulinic acid, is a common in...