Heme Soret Research Articles

CYP8B1 Catalyzes 12alpha-Hydroxylation of C27 Bile Acid: In Vitro Conversion of Dihydroxycoprostanic Acid into Trihydroxycoprostanic Acid.

Sterol 12α-hydroxylase (CYP8B1) is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12α-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12α-hydroxylation of C27 BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12α-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12α-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7α-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12α-hydroxylation of both DHCA and C4, with substrate concentration occupying half of the binding sites of 3.0 and 1.9 μM and kcat of 3.2 and 2.6 minutes-1, respectively. In summary, the confirmed role of CYP8B1 in 12α-hydroxylation of C27 BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. SIGNIFICANCE STATEMENT: The academic community has spent approximately 90 years interpreting the synthesis of bile acids. However, the 12α-hydroxylation of intermediates catalyzed by CYP8B1 is not completely mapped on the classic pathway, particularly for the C27 bile acids, the pivotal intermediates shuttling between mitochondria and peroxisomes. This work discloses the forgotten 12α-hydroxylation pathway from dihydroxycoprostanic acid into trihydroxycoprostanic acid. The present finding may facilitate evaluating CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases.

Dioxygen and glucose force motion of the electron-transfer switch in the iron(III) flavohemoglobin-type nitric oxide dioxygenase

Kinetic and structural investigations of the flavohemoglobin-type NO dioxygenase have suggested critical roles for transient Fe(III)O2 complex formation and O2-forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O2 complex. Stark-effect theory together with structural models and dipole and internal electrostatic field determinations provided a semi-quantitative spectroscopic method for investigating the proposed Fe(III)O2 complex and O2-forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O2 complex. Deoxygenation also elicits Stark effects on the FAD that expose forces and motions that create a more restricted NADH access to FAD for hydride transfer and switch electron-transfer off. Glucose also forces the enzyme toward an off state. Amino acid substitutions at the B10, E7, E11, G8, D5, and F7 positions influence the Stark effects of O2 on resting heme spin states and FAD consistent with the proposed roles of the side chains in the enzyme mechanism. Deoxygenation of ferric myoglobin and hemoglobin A also induces Stark effects on the hemes suggesting a common ‘oxy-met’ state. The ferric myoglobin and hemoglobin heme spectra are also glucose-responsive. A conserved glucose or glucose-6-phosphate binding site is found bridging the BC-corner and G-helix in flavohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O2 storage functions. The results support the proposed roles of a ferric O2 intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover.

Profiling the Cytochrome P450 2J2 Active Site

Human cytochrome P450 2J2 (CYP2J2) is a membrane bound, heme‐containing monooxygenase primary expressed in the heart. This enzyme oxidizes the double bonds in polyunsaturated fatty acids such as arachidonic acid and linoleic acid to epoxides, generating epoxyeicosatrienoic acids (EETs). Various EET products are implicated in cardioprotective effects, but also the promotion of cancer tumor growth and metastasis. Additionally, CYP2J2 can act on a range of pharmaceuticals. Thus, CYP2J2 is of clinical interest, but there is no structural information to understand how the CYP2J2 active site accommodates this wide variety of ligands.In this study, the CYP2J2 active site was defined by its ability to bind an array of >75 substrates, inhibitors, and xenobiotics, including a broad panel of azole compounds. The binding modes and affinities were determined by monitoring the shifts of the CYP2J2 heme Soret absorbance, which is indicative of heme environment perturbation. From these experiments the following spectral shift responses were observed: displacement of the heme‐coordinated water (type‐I binding mode), compound coordination to the heme iron (type‐II), a red‐shifted type‐I binding mode, compound coordination to heme coordinated water (reverse type‐I binding mode), or no spectral shift. As the latter result can be consistent with no binding or binding in the active site in a way that does not perturb the heme center, such compounds were investigated via a luminescence‐based inhibition assay to verify their interaction with CYP2J2. As imidazole‐containing ligands provide a fixed, known reference point for their interaction with the CYP2J2 heme iron, cheminformatics approaches were applied to these compounds to help provide insight into the chemical makeup of the CYP2J2 active site. The physical properties of these ligands were analyzed to identify key ligand features that drive CYP2J2 ligand preference. In the absence of protein structure information, this strategy provides a route to defining the CYP2J2 ligand binding site, potentially supporting the development of selective inhibitors that can help define the role of CYP2J2 modulation in vivo and potentially facilitate therapeutic strategies.

Investigation of Cytochrome P450 2J2 Ligand Binding Modes

Human cytochrome P450 2J2 (CYP2J2) plays a multifaceted role in cardioprotection and xenobiotic metabolism. CYP2J2 oxidizes endogenous polyunsaturated fatty acids P450s, including arachidonic acid and linoleic acid, to various epoxyeicosatrienoic acids (EETs). These EETs are implicated in vasodilation and protection against hypoxia‐reperfusion, but also promote tumor growth and metastasis, where inhibition may be a clinical advantage. Recently, it has been reported that CYP2J2 is inhibited by anti‐hypertensive drugs including manidipine, azelnidipine, and telmisartan. In its drug metabolism role, CYP2J2 metabolizes numerous xenobiotics including anti‐histamine and anti‐cancer agents. While kinetic and metabolic parameters for many of these endogenous and xenobiotic compounds have been reported, it remains to be elucidated how such a wide variety of ligands are accommodated by the CYP2J2 active site. This study determined the binding modes of a catalog of CYP2J2 ligands, including substrates, inhibitors, xenobiotics, and a broad panel of azole containing compounds for the purpose of better understanding their interactions with CYP2J2. The binding modes and affinities were determined by monitoring the spectral shift of the P450 heme Soret absorbance, which is indicative of perturbation of the heme environment. Such spectral shift indicated varying responses: displacement of the heme‐coordinated water (a type‐I binding mode), compound coordination to the heme iron (a type‐II binding mode), or no spectral shift. As the latter result can be consistent with no binding or a binding in the active site in a way that does not perturb the heme center, these latter compounds were investigated via a luminescence‐based inhibition assay to verify their interaction with CYP2J2. Overall, this study aides in improving our understating of different ligand scaffolds that are compatible with CYP2J2 binding to support the development of therapeutics and inhibitors of CYP2J2.Support or Funding InformationNational Institutes of Health Grant R37 GM076343

Design and Synthesis of Imidazole and Triazole Pyrazoles as Mycobacterium Tuberculosis CYP121A1 Inhibitors.

The emergence of untreatable drug‐resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (K D). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert‐butyl (10 g) compounds displaying optimal activity (MIC 1.562 μg/mL, K D 0.22 μM (10 f) and 4.81 μM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H‐bond acceptors/H‐bond donors 4/0, number of rotatable bonds 5–6, molecular volume >340 Å3, topological polar surface area <40 Å2.

Alanine Charge Screening Demonstrates Cytochrome C Unfolding on Cardiolipin Membrane Surfaces

Cytochrome c (Cytc) has been shown to play an important role in signaling during apoptosis. In the initial stages, positively charged regions of Cytc interact electrostatically with negatively charged, cardiolipin (CL) on the inner mitochondrial membrane. In the presence of reactive oxygen species, Cytc demonstrates increased peroxidase activity and a propensity to oxidize CL. Cytc dissociate from the oxidized CL freeing it to form the apoptosome in the cytoplasm and initiate apoptosis. The exact mechanism for this initial electrostatic interaction and the role of structure and conformation of Cytc have been extensively debated. Using heme Soret band circular dichroism (CD), fluorescence (Trp59) and UV-visible spectroscopies, in coordination with alanine charge screening, we investigate the initial electrostatic cytochrome c/cardiolipin interaction.To date, several cardiolipin interaction sites have been proposed on cytochrome c. At pH 8, we are able to isolate and study the cytochrome c/cardiolipin interaction at anionic binding site A with 100% cardiolipin vesicles. Using alanine charge screening (Lys→Ala mutations), we investigated the role of lysines 72, 73, 86 and 87 in site A during electrostatic cardiolipin binding. Optimized titration techniques were utilized that generate reproducible quantitative data that can then be fit to extract the binding contribution to cardiolipin for each amino acid investigated. Our data indicate that an initial binding occurs at lipid to protein, L/P, ratios below 5. However, neither Trp59 fluorescence nor Soret band CD is strongly sensitive to this early binding event making quantitative evaluation of this interaction difficult. At L/P ratios >20 a binding event consistent with a conformational rearrangement on the surface of the CL vesicle occurs. Our data indicate that one lysine from either side of Site A (Lys72/73 versus Lys86/87) is important for both the cooperativity of and L/P ratio needed for this conformational rearrangement on the liposome surface.

Pathways of Transmembrane Electron Transfer in Cytochrome bc Complexes: Dielectric Heterogeneity and Interheme Coulombic Interactions.

The intramembrane cytochrome bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus and the cytochrome b6f complex, which functions in oxygenic photosynthesis, utilize two pairs of b-hemes in a symmetric dimer to accomplish proton-coupled electron transfer. The transmembrane electron transfer pathway in each complex was identified through the novel use of heme Soret band excitonic circular dichroism (CD) spectra, for which the responsible heme-heme interactions were determined from crystal structures. Kinetics of heme reduction and CD amplitude change were measured simultaneously. For bc1, in which the redox potentials of the transmembrane heme pair are separated by 160 mV, heme reduction occurs preferentially to the higher-potential intermonomer heme pair on the electronegative (n) side of the complex. This contrasts with the b6f complex, where the redox potential difference between transmembrane intramonomer p- and n-side hemes is substantially smaller and the n-p pair is preferentially reduced. Limits on the dielectric constant between intramonomer hemes were calculated from the interheme distance and the redox potential difference, ΔEm. The difference in preferred reduction pathway is a consequence of the larger ΔEm between n- and p-side hemes in bc1, which favors the reduction of n-side hemes and cannot be offset by decreased repulsive Coulombic interactions between intramonomer hemes.

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K(m) of 15 ± 4 μM, a k(cat) of 5.2 ± 0.7 min(-1), and a k(cat)/K(m) of 5.8 × 10(3) M(-1) s(-1). From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H2O2, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.

Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

Exciton Interactions Between Hemes bn and bp in the Cytochrome b6f Complex

Circular dichroism spectra have been previously utilized (1) to analyze heme-heme interactions of the two b-hemes in the mitochondrial bc1 complex that were predicted to bridge the ‘B’ and ‘D’ trans-membrane helices on the n- and p-sides of the cytochrome bc complexes (2). It was of interest in the context of the 3.0 A structure of the b6f complex (3-6) and its unique bound chromophoric prosthetic groups, Chl a that is 12 A from heme bp, and heme cn that shares electrons with heme bn, to analyze CD spectra of the heme Soret bands in crystallization-quality b6f complex. Sources of the cytochrome b6f complex were the cyanobacteria, M. laminosus and Nostoc PCC sp. 7120, and spinach thylakoids. In the crystal structures, the oxidized b hemes are separated by 20-21A center-center and 7-8A, edge to edge, and rotated relative to each other by approximately 55° about an axis almost normal to the membrane plane. A bi-lobed dithionite minus ascorbate-reduced CD difference spectrum, qualitatively similar to that seen in the mitochondrial bc1 complex, was obtained from all three sources. Positive and negative bands on the blue and red sides of a 431 nm node, the peak of the absorbance difference spectrum, are diagnostic of excitonic heme-heme interactions. There is no significant contribution to these difference spectra from the Chl a, heme cn, or the heme of cytochrome f.∗deceased; 1, Palmer and Degli-Esposti, 1994; 2, Widger et al. 1984; 3, Kurisu et al. 2003; 4, Stroebel et al. 2003; 5, Yamashita et al. 2007; 6, Baniulis et al. 2009.

Activation of lactoperoxidase by heme‐linked protonation and heme‐independent iodide binding

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.

Assigning Membrane Binding Geometry of Cytochrome c by Polarized Light Spectroscopy

In this work we demonstrate how polarized light absorption spectroscopy (linear dichroism (LD)) analysis of the peptide ultraviolet-visible spectrum of a membrane-associated protein (cytochrome (cyt) c) allows orientation and structure to be assessed with quite high accuracy in a native membrane environment that can be systematically varied with respect to lipid composition. Cyt c binds strongly to negatively charged lipid bilayers with a distinct orientation in which its α-helical segments are on average parallel to the membrane surface. Further information is provided by the LD of the π-π∗ transitions of the heme porphyrin and transitions of aromatic residues, mainly a single tryptophan. A good correlation with NMR data was found, and combining NMR structural data with LD angular data allowed the whole protein to be docked to the lipid membrane. When the redox state of cyt c was changed, distinct variations in the LD spectrum of the heme Soret band were seen corresponding to changes in electronic transition energies; however, no significant change in the overall protein orientation or structure was observed. Cyt c is known to interact in a specific manner with the doubly negatively charged lipid cardiolipin, and incorporation of this lipid into the membrane at physiologically relevant levels was indeed found to affect the protein orientation and its α-helical content. The detail in which cyt c binding is described in this study shows the potential of LD spectroscopy using shear-deformed lipid vesicles as a new methodology for exploring membrane protein structure and orientation.

Force Spectroscopy of the Iron Atom in Heme Proteins

Nuclear resonance vibrational spectroscopy (NRVS) selectively reveals the complete vibrational density of states (VDOS) of a Mossbauer probe nucleus within a protein. Frequency moments of the VDOS determine effective force constants for 57Fe at the active sites of cytochrome c (cyt c) and deoxymyoglobin (Mb). The stiffness measures the force needed to displace the Fe with the other atoms fixed, and probes the nearest neighbor interactions with the Fe. The stiffness of the low spin Fe environment in cyt c greatly exceeds that for the high spin Fe in Mb, reflecting the shorter Fe–N bonds to the heme. Moreover, a significant stiffness decrease upon oxidation of cyt c tracks the longer Fe–S bond to Met 80 in the oxidized protein. Quantitative comparison with 57Fe/54Fe frequency shifts suggests that Fe-L vibrations contribute to the Raman signal of cyt c recorded in resonance with the heme Soret band. The resilience measures the force needed to displace the Fe with the surrounding atoms free to respond, and determines the magnitude of the thermal fluctuations of the Fe on a time scale determined by the experimental energy resolution (ca. 4 ps for the results reported here). Quantitative agreement with the temperature-dependent mean squared displacement determined from independent Mossbauer measurements confirms longstanding assumptions that vibrational motion dominates thermal fluctuations of the heme Fe below the well-known dynamical transition at ca. 200 K and identifies THz frequencies below 100 cm−1 as the dominant contribution. The resilience increases significantly for cyt c with respect to Mb, which we attribute to the increased number of covalent links between heme and peptide in the former protein. Molecular dynamics simulations reproduce the increased resilience of cyt c, but find no significant change with oxidation state.

A One-Residue Switch Reverses the Orientation of a Heme b Cofactor. Investigations of the Ferriheme NO Transporters Nitrophorin 2 and 7 from the Blood-Feeding Insect Rhodnius prolixus

This study represents the identification of a single amino acid residue that has the major responsibility for the isomeric orientation of a heme b cofactor in a ferriheme protein. The insertion of hemin b into the asymmetric environment of a protein pocket facilitates two cofactor orientations, A and B, which is often called "heme rotational disorder". The proteins studied herein are nitrophorins, a class of ferriheme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus, in this case NP2 and NP7. NMR spectroscopy (pH* 5.5) of the imidazole complex of NP7 revealed solely the A orientation, whereas NP2 shows primarily the B orientation ( approximately 1:5 A:B). The glutamate 27 residue in NP7 is an obvious difference in the heme pocket compared to those of NP1-4, all of which present a valine residue [valine 24 (NP2 and NP3) or valine 25 (NP1 and NP4)] at the same position. Consequently, the mutant NP2(V24E) was prepared and shown to reverse the heme orientation to exclusively A, whereas NP7(E27V) revealed an approximately 1:3 A:B ratio. The reversal A <--> B following the change glutamine <--> valine was further indicated in circular dichroism (CD) spectroscopy with a positive (A) or negative (B) Deltaepsilon of the heme Soret band. Moreover, CD spectroscopy was applied to the mutant NP7(E27Q) and indicated mainly the A orientation, which allows us to conclude that the steric hindrance provided by the glutamate residue is responsible for the heme orientation rather then the carboxylate charge.

Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)

Cytochrome P450 170A1 (CYP170A1) is encoded by the sco5223 gene of the Gram-positive, soil-dwelling bacterium Streptomyces coelicolor A3(2) as part of a two-gene cluster with the sco5222 gene. The SCO5222 protein is a sesquiterpene synthase that catalyzes the cyclization of farnesyl diphosphate to the novel tricyclic hydrocarbon, epi-isozizaene (Lin, X., Hopson, R., and Cane, D. E. (2006) J. Am. Chem. Soc. 128, 6022-6023). The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wild-type strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2: HIGHLY COOPERATIVE PYRENE HYDROXYLATION AND MULTIPHASIC KINETICS OF LIGAND BINDING

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of alpha-naphthoflavone (alphaNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for alphaNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (alphaNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-alphaNF complex show active site space for only one alphaNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.

Ligand Binding to Cytochrome P450 3A4 in Phospholipid Bilayer Nanodiscs: THE EFFECT OF MODEL MEMBRANES

The membrane-bound protein cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme. Most studies of ligand binding by CYP3A4 are currently carried out in solution, in the absence of a model membrane. Therefore, there is little information concerning the membrane effects on CYP3A4 ligand binding behavior. Phospholipid bilayer Nanodiscs are a novel model membrane system derived from high density lipoprotein particles, whose stability, monodispersity, and consistency are ensured by their self-assembly. We explore the energetics of four ligands (6-(p-toluidino)-2-naphthalenesulfonic acid (TNS), alpha-naphthoflavone (ANF), miconazole, and bromocriptine) binding to CYP3A4 incorporated into Nanodiscs. Ligand binding to Nanodiscs was monitored by a combination of environment-sensitive ligand fluorescence and ligand-induced shifts in the fluorescence of tryptophan residues present in the scaffold proteins of Nanodiscs; binding to the CYP3A4 active site was monitored by ligand-induced shifts in the heme Soret band absorbance. The dissociation constants for binding to the active site in CYP3A4-Nanodiscs were 4.0 microm for TNS, 5.8 microm for ANF, 0.45 microm for miconazole, and 0.45 microm for bromocriptine. These values are for CYP3A4 incorporated into a lipid bilayer and are therefore presumably more biologically relevant that those measured using CYP3A4 in solution. In some cases, affinity measurements using CYP3A4 in Nanodiscs differ significantly from solution values. We also studied the equilibrium between ligand binding to CYP3A4 and to the membrane. TNS showed no marked preference for either environment; ANF preferentially bound to the membrane, and miconazole and bromocriptine preferentially bound to the CYP3A4 active site.

YcdB from Escherichia coli Reveals a Novel Class of Tat-dependently Translocated Hemoproteins

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe(III)-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.

C-terminal Tail Residue Arg1400 Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-oxide Synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.

Luminescent Ruthenium(II)− and Rhenium(I)−Diimine Wires Bind Nitric Oxide Synthase

Ru(II)- and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)2L']2+, L' is a perfluorinated biphenyl bridge connecting 4,4'-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Delta65 and Delta114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Delta65 and Delta114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru-diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO)3L1L1']+, where L1 is 4,7-dimethylphenanthroline and L1' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F8bp-im) (4) or F (F9bp) (5), also form complexes with Delta114. Binding of 4 shifts the Delta114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Delta114 dissociation constant is 100 +/- 80 nM. Re-wire 5 binds Delta114 with a K(d) of 5 +/- 2 microM, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe.