FMN Domain Research Articles

Reaction of Human Cytochrome P450 3A4 with Peroxynitrite: Nitrotyrosine Formation on the Proximal Side Impairs Its Interaction with NADPH-Cytochrome P450 Reductase

The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.

Control of Electron Transfer and Catalysis in Neuronal Nitric-oxide Synthase (nNOS) by a Hinge Connecting Its FMN and FAD-NADPH Domains

In nitric-oxide synthases (NOSs), two flexible hinges connect the FMN domain to the rest of the enzyme and may guide its interactions with partner domains for electron transfer and catalysis. We investigated the role of the FMN-FAD/NADPH hinge in rat neuronal NOS (nNOS) by constructing mutants that either shortened or lengthened this hinge by 2, 4, and 6 residues. Shortening the hinge progressively inhibited electron flux through the calmodulin (CaM)-free and CaM-bound nNOS to cytochrome c, whereas hinge lengthening relieved repression of electron flux in CaM-free nNOS and had no impact or slowed electron flux through CaM-bound nNOS to cytochrome c. How hinge length influenced heme reduction depended on whether enzyme flavins were pre-reduced with NADPH prior to triggering heme reduction. Without pre-reduction, changing the hinge length was deleterious; with pre-reduction, the hinge shortening was deleterious, and hinge lengthening increased heme reduction rates beyond wild type. Flavin fluorescence and stopped-flow kinetic studies on CaM-bound enzymes suggested hinge lengthening slowed the domain-domain interaction needed for FMN reduction. All hinge length changes lowered NO synthesis activity and increased uncoupled NADPH consumption. We conclude that several aspects of catalysis are sensitive to FMN-FAD/NADPH hinge length and that the native hinge allows a best compromise among the FMN domain interactions and associated electron transfer events to maximize NO synthesis and minimize uncoupled NADPH consumption.

Nitric oxide synthases in motion

To determine a dynamic functional model of full-length nitric oxide synthase (NOS), we are coupling Small Angle X-ray Scattering (SAXS) with our crystal structures of individual NOS components. SAXS is a solution scattering method that is well suited to capturing protein conformational changes. Our lab has solved crystal structures of all three parts of NOS enzymes: NOS oxygenase, NOS reductase and NOS calmodulin (CaM) peptide with CaM bound. Based on our crystal structures and biochemical data, we propose that the NOS FMN domain swings from the electron-accepting “IN” to an electron-donating “OUT” conformation. In the electron-accepting conformation observed in our crystal structure, the FMN is buried and positioned to accept an electron from redox partner FAD. We propose that the entire FMN domain swings “OUT” from the remainder of the reductase domain to transfer electrons to the active site heme. We are combining scattering data with atomic resolution crystal structures and molecular dynamics simulations to identify snapshots of NOS in solution. We analyze and compare FMN domain movement in different isozymes, making use of overlapping truncated constructs and post-translationally modified proteins. Our SAXS results are consistent with a large swinging movement in the FMN domain that could enable electron transfer from FMN to heme.

Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BackgroundHuman endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear.Methodology/Principal FindingsUsing a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2–3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS.ConclusionsOur results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding.

Control Of Electron Transfer And Catalysis In Neuronal NOS By A Hinge Connecting The FMN And FNR Domains

In nitric oxide synthase enzymes (NOSs) two flexible hinge elements connect the FMN domain to the rest of the enzyme and may guide FMN domain interactions with its partner FNR and NOSoxy domains to enable the electron transfers required during catalysis. In our previous study, we found that incorporating the nNOS FMN‐FNR hinge into eNOS increased its NO synthesis activity 4‐fold, decreased uncoupled NADPH oxidation, and supported faster heme reduction (kr), implying that the composition and/or length of the FMN‐FNR hinge is important for determining electron transfer and catalysis. To more systematically investigate the role of the FMN‐FNR hinge we constructed mutants in nNOS that either shorten or lengthen the hinge by 2, 4, and 6 residues. We found that FMN‐FNR hinge length helps determine electron flux through the nNOS flavoprotein domain. Shortening the hinge inhibited electron flux through both the calmodulin (CaM)‐free and CaM‐bound enzymes, while lengthening the hinge relieved the repression on electron flux that is normally present in the CaM‐free enzyme, and has either no impact or was deleterious for electron flux through the CaM‐bound enzyme. The nNOS kr was also sensitive to FMN‐FNR hinge length. Small changes in hinge length were tolerated, while changes beyond 2 or 4 residues were deleterious to kr, and also decoupled nNOS NADPH consumption from NO synthesis. Supported by NIH GM51941 & HL63744.

Fluorescence detection of obligatory conformations in catalysis and control of NOS

Nitric Oxide synthases produce NO as a signal and as a cytotoxin in immune response. In the tethered shuttle model for NOS reductase kinetics, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic sites. FMN fluorescence is sensitive to NOS conformation; eNOS and nNOS holoenzymes have states with lifetimes of ~4.3ns, 1 ns, and 90 ps. Two domain oxyFMN constructs (truncated before the FAD binding domain) and holoenzyme share open conformations with lifetimes of ~4.3 ns. The holoenzyme majority state has a lifetime of ~90 ps due to FAD/FMN proximity as in crystal structures (Wang et al, Garcin et al). In oxyFMN ~25% of FMN has a lifetime of 0.9 ns, which we attribute to quenching by heme. Calmodulin activation of NOS releases the FMN binding domain from the ‘dehydrogenase’ complex, increasing the populations of the long lifetime states. Calmodulin binding shortens the average lifetime of oxyFMN constructs, because CaM promotes association of the FMN binding domain with the oxygenase domain, and favors the 1 ns state over the 4.3 ns state. Similar effects help explain modulation of NOS activity by covalent modification and protein‐protein interactions, e.g., in the HSP90‐eNOS complex.

FMN fluorescence in inducible NOS constructs reveals a series of conformational states involved in the reductase catalytic cycle

Nitric oxide synthases (NOSs) produce NO as a molecular signal in the nervous and cardiovascular systems and as a cytotoxin in the immune response. NO production in the constitutive isoforms is controlled by calmodulin regulation of electron transfer. In the tethered shuttle model for NOS reductase function, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic centers. Crystal structures of neuronal NOS reductase domain and homologs correspond to an 'input state', with FMN in close contact with FAD. We recently produced two domain 'output state' (oxyFMN) constructs showing calmodulin dependent FMN domain association with the oxygenase domain. FMN fluorescence is sensitive to enzyme conformation and calmodulin binding. The inducible NOS (iNOS) oxyFMN construct is more fluorescent than iNOS holoenzyme. The difference in steady state fluorescence is rationalized by the observation of a series of characteristic states in the two constructs, which we assign to FMN in different environments. OxyFMN and holoenzyme share open conformations with an average lifetime of ~4.3 ns. The majority state in holoenzyme has a short lifetime of ~90 ps, probably because of FAD-FMN interactions. In oxyFMN about 25-30% of the FMN is in a state with a lifetime of 0.9 ns, which we attribute to quenching by heme in the output state. Occupancy of the output state together with our previous kinetic results yields a heme edge to FMN distance estimate of 12-15 Å. These results indicate that FMN fluorescence is a valuable tool to study conformational states involved in the NOS reductase catalytic cycle.

The crystal structure of the FAD/NADPH‐binding domain of flavocytochrome P450 BM3

We report the crystal structure of the FAD/NADPH-binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP(+), identifying important protein interactions with the NADPH 2'-phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH-dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild-type FAD domain to be near-exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme's connecting domain (that links the FAD domain to the FMN-binding domain in P450 BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin-like) domain within the BM3 reductase module.

Role of an isoform-specific serine residue in FMN–heme electron transfer in inducible nitric oxide synthase

In the crystal structure of a calmodulin (CaM)-bound FMN domain of human inducible nitric oxide synthase (NOS), the CaM-binding region together with CaM forms a hinge, and pivots on an R536(NOS)/E47(CaM) pair (Xia et al. J Biol Chem 284:30708-30717, 2009). Notably, isoform-specific human inducible NOS S562 and C563 residues form hydrogen bonds with the R536 residue through their backbone oxygens. In this study, we investigated the roles of the S562 and C563 residues in the NOS FMN-heme interdomain electron transfer (IET), the rates of which can be used to probe the interdomain FMN/heme alignment. Human inducible NOS S562K and C563R mutants of an oxygenase/FMN (oxyFMN) construct were made by introducing charged residues at these sites as found in human neuronal NOS and endothelial NOS isoforms, respectively. The IET rate constant of the S562K mutant is notably decreased by one third, and its flavin fluorescence intensity per micromole per liter is diminished by approximately 24%. These results suggest that a positive charge at position 562 destabilizes the hydrogen-bond-mediated NOS/CaM alignment, resulting in slower FMN-heme IET in the mutant. On the other hand, the IET rate constant of the C563R mutant is similar to that of the wild-type, indicating that the mutational effect is site-specific. Moreover, the human inducible NOS oxyFMN R536E mutant was constructed to disrupt the bridging CaM/NOS interaction, and its FMN-heme IET rate was decreased by 96%. These results demonstrated a new role of the isoform-specific serine residue of the key CaM/FMN(NOS) bridging site in regulating the FMN-heme IET (possibly by tuning the alignment of the FMN and heme domains).

Comparing the temperature dependence of FMN to heme electron transfer in full length and truncated inducible nitric oxide synthase proteins

The FMN-heme interdomain (intraprotein) electron transfer (IET) kinetics in full length and oxygenase/FMN (oxyFMN) construct of human iNOS were determined by laser flash photolysis over the temperature range from 283 to 304K. An appreciable increase in the rate constant value was observed with an increase in the temperature. Our previous viscosity study indicated that the IET process is conformationally gated, and Eyring equation was thus used to analyze the temperature dependence data. The obtained magnitude of activation entropy for the IET in the oxyFMN construct is only one-fifth of that for the holoenzyme. This indicates that the FMN domain in the holoenzyme needs to sample more conformations before the IET takes place, and that the FMN domain in the oxyFMN construct is better poised for efficient IET.

A kinetic model linking protein conformational motions, interflavin electron transfer and electron flux through a dual‐flavin enzyme – simulating the reductase activity of the endothelial and neuronal nitric oxide synthase flavoprotein domains

NADPH-dependent dual-flavin enzymes provide electrons in many redox reactions, although the mechanism responsible for regulating their electron flux remains unclear. We recently proposed a four-state kinetic model that links the electron flux through a dual-flavin enzyme to its rates of interflavin electron transfer and FMN domain conformational motion [Stuehr DJ et al. (2009) FEBS J276, 3959-3974]. In the present study, we ran computer simulations of the kinetic model to determine whether it could fit the experimentally-determined, pre-steady-state and steady-state traces of electron flux through the neuronal and endothelial NO synthase flavoproteins (reductase domains of neuronal nitric oxide synthase and endothelial nitric oxide synthase, respectively) to cytochrome c. We found that the kinetic model accurately fitted the experimental data. The simulations gave estimates for the ensemble rates of interflavin electron transfer and FMN domain conformational motion in the reductase domains of neuronal nitric oxide synthase and endothelial nitric oxide synthase, provided the minimum rate boundary values, and predicted the concentrations of the four enzyme species that cycle during catalysis. The findings of the present study suggest that the rates of interflavin electron transfer and FMN domain conformational motion are counterbalanced such that both processes may limit electron flux through the enzymes. Such counterbalancing would allow a robust electron flux at the same time as keeping the rates of interflavin electron transfer and FMN domain conformational motion set at relatively slow levels.

Pulsed ENDOR determination of relative orientation of g-frame and molecular frame of imidazole-coordinated heme center of iNOS.

Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe-FMN radius vector, R(Fe-FMN), with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g-frame and molecular frame, from which R(Fe-FMN) can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, whereas the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works.

Effect of solution viscosity on intraprotein electron transfer between the FMN and heme domains in inducible nitric oxide synthase

The FMN–heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN construct were determined by laser flash photolysis as a function of solution viscosity (1.0–3.0cP). In the presence of ethylene glycol or sucrose, an appreciable decrease in the IET rate constant value was observed with an increase in the solution viscosity. The IET rate constant is inversely proportional to the viscosity for both viscosogens. This demonstrates that viscosity, and not other properties of the added viscosogens, causes the dependence of IET rates on the solvent concentration. The IET kinetics results indicate that the FMN–heme IET in iNOS is gated by a large conformational change of the FMN domain. The kinetics and NOS flavin fluorescence results together indicate that the docked FMN/heme state is populated transiently.

Mutation in the Flavin Mononucleotide Domain Modulates Magnetic Circular Dichroism Spectra of the iNOS Ferric Cyano Complex in a Substrate-Specific Manner

We have obtained low-temperature magnetic circular dichroism (MCD) spectra for ferric cyano complexes of the wild type and E546N mutant of a human inducible nitric oxide synthase (iNOS) oxygenase/flavin mononucleotide (oxyFMN) construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the l-arginine-bound ferric iNOS heme (Sempombe, J.; et al. J. Am. Chem. Soc. 2009, 131, 6940-6941). The addition of l-arginine to the wild-type protein causes notable changes in the CN(-)-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with l-arginine is absent in the CN(-) complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. These results indicate that interdomain FMN-heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.

Homology modeling of the three membrane proteins of the dhurrin metabolon: Catalytic sites, membrane surface association and protein–protein interactions

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein–protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F′ and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase

CPR (NADPH-cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.

Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes

Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His 179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure.

Electron transfer in a human inducible nitric oxide synthase oxygenase/FMN construct co-expressed with the N-terminal globular domain of calmodulin

The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1–75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca 2+ depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca 2+-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET.

Cloning, Expression and Purification of Cindoxin, an Unusual Fmn‐Containing Cytochrome P450 Redox Partner

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450(cin) (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6<pH<8; this enabled the quinone/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) redox potentials to be determined. Our results show that cindoxin might be somewhat different to other flavodoxins that interact with P450s, in which generally only one couple is important. Both redox states of cindoxin could be catalytically relevant. A catalytically active system was reconstituted in vitro with E. coli flavodoxin reductase (Fpr) acting as the terminal redox partner in the absence of CdR. Our results show that Cdx and Fpr support regio- and stereoselective P450(cin)-catalysed cineole oxidation to (1R)-6beta-hydroxycineole with turnover rates up to 1500 min(-1). This system is tightly coupled with 80 % of NADPH reducing equivalents funnelled into substrate oxidation.