Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

Jenkins, Christopher M.; Genzor, Carlos G.; Fillat, Marı́a F.; Waterman, Michael R.; Gómez‐Moreno, Carlos

doi:10.1074/jbc.272.36.22509

Cited by 31 publications

(24 citation statements)

References 38 publications

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“…The mutation did not affect cytochrome c or ferricyanide reductase activities, indicating interaction between the reductase and these molecules is distinct from its interaction with cytochrome P450. Similar observations were reported in Anabaena flavodoxin (29), where mutagenic analysis of cluster I acidic residues Asp 144 and Glu 145 showed they were involved in flavodoxin-supported P450c 17 progesterone 17a-hydroxylase activity but not involved in cytochrome c reduction. Crystal structures of cytochrome P450 reductase (17) and Anabaena flavodoxin (30) show that cluster 1 residues are located near the surface, presumably positioned to interact with a positive surface patch on their hemeprotein acceptor.…”

Section: Synthesis Of Nitric Oxide (No)supporting

confidence: 71%

“…Thus, cluster 1 may function differently in nNOS as compared with other flavoproteins of its class. Crystal structures of cytochrome P450 reductase and Anabaena flavodoxin show that cluster 1 acidic residues and the conserved Phe (analogous to nNOS Phe 892 ) are positioned near the surface of the FMN module and away from the bound FMN (17,29). If nNOS cluster 1 residues are similarly positioned, they would need to influence FMN binding through relatively long-range interactions.…”

Section: Characterization Of Fmn-free Neuronal No Synthasementioning

confidence: 99%

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Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

Adak

Ghosh

Abu-Soûd

et al. 1999

Journal of Biological Chemistry

137

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The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp 918 and Glu 919 ) and one conserved aromatic residue (Phe 892 ), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steadystate and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.

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Section: Synthesis Of Nitric Oxide (No)supporting

confidence: 71%

Section: Characterization Of Fmn-free Neuronal No Synthasementioning

confidence: 99%

Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

Adak

Ghosh

Abu-Soûd

et al. 1999

Journal of Biological Chemistry

137

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“…However, the rate is considerably elevated by the presence of E. coli FLD. In a recent publication, Jenkins and coworkers [19] reported the kinetics of cytochrome c reduction with the Anabaena variabilis flavodoxin NADP ϩ reductase/flavodoxin system. In this system, there is negligible flavodoxinindependent cytochrome c reduction by the Anabaena FLDR.…”

Section: Discussionmentioning

confidence: 99%

Characterisation of flavodoxin NADP⁺ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli

McIver

Leadbeater

Campopiano

et al. 1998

European Journal of Biochemistry

103

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The genes encoding the Escherichia coli flavodoxin NADP ϩ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a k cat of 1610.3 min Ϫ1 and a K m of 23.6 µM, and cytochrome c with a k cat of 141.3 min Ϫ1 and a K m of 17.6 µM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent K m of 6.84 µM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isolectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (Ϫ308 mV and Ϫ268 mV, respectively) and FLD (Ϫ254 mV and Ϫ433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH→FLDR→FLD. Binding of 2′ adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M Ϫ1 cm Ϫ1 at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.Keywords : flavodoxin ; flavodoxin NADP ϩ oxidoreductase; redox potentiometry; enzyme kinetics; cytochrome P-450.The Escherichia coli flavodoxin NADP ϩ oxidoreductase (FLDR or flavodoxin reductase) and flavodoxin (FLD) are the two flavin-containing components of a short electron-transfer chain from NADPH, which provides electrons for the function of the biotin synthase [1] and cobalamin-dependent methionine synthase systems [2]. The enzymes are also required during anaerobic growth of the organism, participating in the pyruvate formate/lyase system of E. coli Ϫ a crucial mechanism for the anaerobic generation of pyruvate for glycolysis [3] and in the generation of deoxyribonucleotides through the enzyme anaerobic ribonucleotide reductase [4]. Recently, the FLDR/FLD system has also been recognised as the E. coli 'reductase', which can support the functio...

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“…Thus, Lys75, Leu76, and Leu78 in FNR are also essential in the stabilization of an optimal complex for ET between FNR and Fld (7,(17)(18)(19). However, although the roles of several Fld residues, either acidic or hydrophobic in nature (namely, Trp57, Ile59, Glu61, Glu67, Ile92, Tyr94, Asp100, Asp126, Asp144, and Glu145), have been analyzed, none of them has shown to be critical neither for complex formation nor for ET between Fld and FNR (19)(20)(21)(22). Thus, the two aromatic residues, Trp57 and Tyr94, that sandwich the FMN cofactor do not play an active role in the Fld redox reactions with FNR.…”

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confidence: 99%

Anabaena Flavodoxin as an Electron Carrier from Photosystem I to Ferredoxin-NADP⁺ Reductase. Role of Flavodoxin Residues in Protein−Protein Interaction and Electron Transfer

et al. 2004

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Biochemical and structural studies indicate that electrostatic and hydrophobic interactions are critical in the formation of optimal complexes for efficient electron transfer (ET) between ferredoxin-NADP + reductase (FNR) and ferredoxin (Fd). Moreover, it has been shown that several charged and hydrophobic residues on the FNR surface are also critical for the interaction with flavodoxin (Fld), although, so far, no key residue on the Fld surface has been found to be the counterpart of such FNR side chains. In this study, negatively charged side chains on the Fld surface have been individually modified, either by the introduction of positive charges or by their neutralization. Our results indicate that although Glu16, Glu20, Glu61, Asp65, and Asp96 contribute to the orientation and optimization of the Fld interaction, either with FNR or with photosystem I (PSI) (presumably through the formation of salt bridges), for efficient ET, none of these side chains is involved in the formation of crucial salt bridges for optimal interaction with FNR. These data support the idea that the FNR-Fld interaction is less specific than the FNR-Fd interaction. However, analysis of the reactivity of these mutated Flds toward the membraneanchored PSI complex indicated that all mutants, except Glu16Gln, lack the ability to form a stable complex with PSI. Thr12, Thr56, Asn58, and Asn97 are present in the close environment of the isoalloxazine ring of FMN in Anabaena Fld. Their roles in the interaction with and ET to FNR and PSI have also been studied. Mutants at these Fld positions indicate that residues in the close environment of the isoalloxazine ring modulate the ability of Fld to bind to and to exchange electrons with its physiological counterparts.Electrostatic and hydrophobic interactions play an important role in the formation of optimal complexes for efficient electron transfer (ET) 1 between proteins (1, 2). In the photosynthetic ET chain, electrons are transferred from photosystem I (PSI) to ferredoxin-NADP + reductase (FNR) via ferredoxin (Fd). In this system, specific recognition and binding between FNR and Fd are required for efficient ET (3-7). Thus, electrostatic interactions orient the proteins for formation of an initial complex, whereas hydrophobic interactions are critical in the formation of the optimal complex for ET (5,6,8). The structures of the FNR-Fd complexes from Anabaena and maize have been determined by X-ray crystallography (9, 10), confirming the importance of the nature of the interactions that was established by biochemical studies. Thus, biochemical and structural data indicate that Fd binds in a concave cavity around the FAD group of the reductase, where residues Lys75, Leu76, and Leu78 on the FNR surface play a crucial role in complex formation, by interacting with the side chains of Glu94 and Phe65 on [11][12][13][14].Flavodoxins (Fld) are small R/ flavoproteins that contain a noncovalently bound FMN cofactor. In cyanobacteria and certain algae, Fld is synthesized instead of Fd when the or...

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Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

Cited by 31 publications

References 38 publications

Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

Characterisation of flavodoxin NADP⁺ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli

Anabaena Flavodoxin as an Electron Carrier from Photosystem I to Ferredoxin-NADP⁺ Reductase. Role of Flavodoxin Residues in Protein−Protein Interaction and Electron Transfer

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Negatively Charged Anabaena Flavodoxin Residues (Asp144 and Glu145) Are Important for Reconstitution of Cytochrome P450 17α-Hydroxylase Activity

Cited by 31 publications

References 38 publications

Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

Role of Reductase Domain Cluster 1 Acidic Residues in Neuronal Nitric-oxide Synthase

Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli

Anabaena Flavodoxin as an Electron Carrier from Photosystem I to Ferredoxin-NADP+ Reductase. Role of Flavodoxin Residues in Protein−Protein Interaction and Electron Transfer

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Characterisation of flavodoxin NADP⁺ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli

Anabaena Flavodoxin as an Electron Carrier from Photosystem I to Ferredoxin-NADP⁺ Reductase. Role of Flavodoxin Residues in Protein−Protein Interaction and Electron Transfer