Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5å resolution: relationship to other flavoprotein reductases

Lu, Guoguang; Campbell, Wilbur H.; Schneider, Günter; Lindqvist, Ylva

doi:10.1016/s0969-2126(94)00082-4

Cited by 90 publications

(82 citation statements)

References 42 publications

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“…Although CbR has two domains, one for binding NADH and one for binding FAD, the linker joining them is tightly associated with the NADH domain and is not considered a flexible hinge (1,6,7). MoR adds the heme-Fe or Cyt b domain to CbR via the flexible Hinge 2 of holo-NaR ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Barbier¹,

Campbell²

2005

Journal of Biological Chemistry

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Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD-(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.

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Section: Resultsmentioning

confidence: 99%

“…For CbR containing only the FAD cofactor, the absorbance at 460 nm was measured and an extinction coefficient of 10 mM Ϫ1 cm Ϫ1 was used (6,11).…”

Section: Methodsmentioning

confidence: 99%

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Barbier¹,

Campbell²

2005

Journal of Biological Chemistry

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“…To gain a better understanding of the influence of mAb NL7 binding on oxidase activity, we modeled most of the C-terminal domain of gp91 phox (amino acid residues 291 S-F 565 ) to the known crystal structure of a related protein, corn nitrate reductase (pdb 1CNF) (30). In the space-filling and ribbon images showing the crystal structure of corn nitrate reductase (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We conclude that the 498 EKDVITGLK 506 domain of FIGURE 7. Structural model comparing the crystal structure of 1CNF (30) to the predicted carboxyl portion of gp91 phox . A and C, The 1CNF structure is shown in space-filling and ribbon rendition, respectively.…”

Section: Discussionmentioning

confidence: 99%

Functional Epitope on Human Neutrophil Flavocytochrome b558

Burritt

Foubert

Baniulis

et al. 2003

The Journal of Immunology

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mAb NL7 was raised against purified flavocytochrome b558, important in host defense and inflammation. NL7 recognized the gp91phox flavocytochrome b558 subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the 498EKDVITGLK506 region of gp91phox. In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (Km). mAb NL7 did not inhibit translocation of p47phox, p67phox, or Rac to the plasma membrane, and bound its epitope on gp91phox independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91phox on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47phox binding sites. We conclude that the 498EKDVITGLK506 segment resides on the cytosolic surface of gp91phox and represents a region important for oxidase function, but not substrate or cytosolic component binding.

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“…with other members of the ferredoxin reductase family of enzymes, including spinach ferredoxin-NADP + oxidoreductase (FNR) [10], corn nitrate reductase [11] and porcine cytochrome b & reductase [12]. The FLDR structure reveals an unusual flavinbinding conformation, with a hydrogen bond between the adenine and isoalloxazine rings, possibly explaining the unusual visible spectrum of the bound flavin [7].…”

Section: Introductionmentioning

confidence: 99%

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

Leadbeater

McIver

Campopiano

et al. 2000

Biochemical Journal

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The structure of the Escherichia coli flavodoxin NADP + oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min V " ; R174A, 132.1 min V " ; R184A, 305.5 min V " versus wild type, 338.9 min V ") and increased K m for NADPH (R144A, 5.3 µM ; R174A, 20.2 µM ; R184A, 54.4 µM versus wild type, 3.9 µM). The k cat value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min V ") and R184A (50.4 min V ") compared with the wild type (33.0 min V "), consistent with roles for R174 and R184 in discriminating between NADPH\NADH by interaction with the adenosine ribose 2h-phosphate. Stopped-flow studies indicated that affinity (K d ) for NADPH was markedly reduced in mutants R144A (635 µM) and R184A (2.3 mM) compared with the wild

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Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5å resolution: relationship to other flavoprotein reductases

Cited by 90 publications

References 42 publications

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

Functional Epitope on Human Neutrophil Flavocytochrome b558

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

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