Functional selectivity in cannabinoid signaling.

Cannabinoid (CB) agonists exhibit numerous potentially useful pharmacological properties, but unwanted side effects limit their use in clinical practice. Thus, novel strategies are needed to identify potential CB pharmaceuticals with fewer side effects. Activated CB receptors initiate multiple parallel intracellular signal transduction cascades. In the present paper we will review experimental data indicating that structurally different classes of CB agonists may exhibit selectivity toward individual subsets of intracellular signaling pathways. In support of this, recent findings indicate that chemically distinct classes of CB agonists frequently differ in their rank order of potency to produce analgesia versus other central nervous system effects in vivo. Structurally different agonists were also found to differ in their abilities to activate individual G protein types in vitro. Since it was suggested earlier that structurally distinct CB agonists may interact differently with the CB receptors, it has been hypothesized that different classes of cannabinoid agonists may stabilize unique active CB receptor conformations, leading to functional selectivity in CB receptor signaling. In order to obtain a direct proof for this hypothesis, we recently employed a highly sensitive biophysical method, plasmon-waveguide resonance (PWR) spectroscopy. PWR experiments have provided a direct proof that structurally different CB agonists produce qualitatively distinct changes in the shape and/or membrane orientation of the CB1 receptors, leading to functional selectivity in G protein activation. We expect that by identification of CB agonists that selectively activate preferred intracellular signaling pathways novel pharmacological lead structures can be identified for the design of improved CB analgesics with fewer side effects.

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein.

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.

Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy.

The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.

Rhodobacter capsulatus photoactive yellow protein: genetic context, spectral and kinetics characterization, and mutagenesis.

A gene for photoactive yellow protein (PYP) was previously cloned from Rhodobacter capsulatus (Rc), and we have now found it to be associated with genes for gas vesicle formation in the recently completed genome sequence. However, the PYP had not been characterized as a protein. We have now produced the recombinant RcPYP in Escherichia coli as a glutathione-S-transferase (GST) fusion protein, along with the biosynthetic enzymes, resulting in the formation of holo-RcPYP following cleavage of the GST tag. The absorption spectrum (with characteristic peaks at 435 and 375 nm) and the photocycle kinetics, initiated by a laser flash at 445 nm, are generally similar to those of Rhodobacter sphaeroides (RsPYP) but are significantly different from those of the prototypic PYP from Halorhodospira halophila (HhPYP), which has a single peak at 446 nm and has slower recovery. RcPYP also is photoactive when excited with near-ultraviolet laser light, but the end point is then above the preflash baseline. This suggests that some of the PYP chromophore is present in the cis-protonated conformation in the resting state. The excess 435 nm form in RcPYP, built up from repetitive 365 nm laser flashes, returns to the preflash baseline with an estimated half-life of 2 h, which is markedly slower than that for the same reaction in RsPYP. Met100 has been reported to facilitate cis-trans isomerization in HhPYP, yet both Rc and RsPYPs have Lys and Gly substitutions at positions 99 and 100 (using HhPYP numbering throughout) and have 100-fold faster recovery kinetics than does HhPYP. However, the G100M and K99Q mutations of RcPYP have virtually no effect on kinetics. Apparently, the RcPYP M100 is in a different conformation, as was recently found for the PYP domain of Rhodocista centenaria Ppr. The cumulative results show that the two Rhodobacter PYPs are clearly distinct from the other species of PYP that have been characterized. These properties also suggest a different functional role, that we postulate to be in regulation of gas vesicle genes, which are known to be light-regulated in other species.

Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila.

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.

Binding of agonists, antagonists and inverse agonists to the human delta-opioid receptor produces distinctly different conformational states distinguishable by plasmon-waveguide resonance spectroscopy.

Structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned delta-opioid receptor from human brain immobilized in a solid-supported lipid bilayer were monitored using plasmon-waveguide resonance (PWR) spectroscopy. Agonist (e.g. deltorphin II) binding causes an increase in membrane thickness because of receptor elongation, a mass density increase due to an influx of lipid molecules into the bilayer, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (e.g. TIPPpsi) binding produces no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter thought to be due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) [Salamon et al. (2000) Biophys. J.79, 2463-2474]. In addition, we now find that an inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand-induced G-protein-coupled receptor functioning. The potential utilization of this new biophysical method to examine structural changes both parallel and perpendicular to the membrane normal for GPCRs is emphasized.

Early photocycle kinetic behavior of the E46A and Y42F mutants of photoactive yellow protein: femtosecond spectroscopy.

In the photoactive yellow protein, PYP, both Glu46 and Tyr42 form hydrogen bonds to the phenolic OH group of the p-hydroxycinnamoyl chromophore. Previous work on replacement of the carboxyl group of Glu46 by an amide group (Glu46Gln) has shown that changing the nature of this hydrogen bond has a minimal effect on the rate constant for the formation of the first intermediate (I(0)) and on the excited state lifetime, whereas the rate constants for the formation of the second (I(0)( not equal)) and third (I(1)) intermediates were increased by factors of approximately 30 and 5, respectively. In the present experiments, two additional mutants (Glu46Ala and Tyr42Phe) have been studied. These two mutants are shown to behave kinetically very similarly to one another. In both cases, the rate constant for I(0) formation is decreased by a factor of approximately 2, with little or no effect on the photochemical yield as a consequence of a compensating increase in the excited state lifetime. Although we are unable to resolve the rate constant for the formation of the second intermediate from that of the first intermediate, the rate constant for the formation of the third intermediate is increased by a factor of approximately 100. The structural implications of these results are discussed.

Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction.

The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam. The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system. We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx. Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)). The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin. The rate of FADH(*) decay was dependent on ionic strength and NAD(+). In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed. The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively. This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase. Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.

Role of a cluster of hydrophobic residues near the FAD cofactor in Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal complex formation and electron transfer to ferredoxin.

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.

Optical anisotropy in lipid bilayer membranes: coupled plasmon-waveguide resonance measurements of molecular orientation, polarizability, and shape.

The birefringence and linear dichroism of anisotropic thin films such as proteolipid membranes are related to molecular properties such as polarizability, shape, and orientation. Coupled plasmon-waveguide resonance (CPWR) spectroscopy is shown in the present work to provide a convenient means of evaluating these parameters in a single lipid bilayer. This is illustrated by using 1-10 mol % of an acyl chain chromophore-labeled phosphatidylcholine (PC) incorporated into a solid-supported PC bilayer deposited onto a hydrated silica surface. CPWR measurements were made of refractive index and extinction coefficient anisotropies with two exciting light wavelengths, one of which is absorbed by the chromophore and one of which is not. These results were used to calculate longitudinal and transverse molecular polarizabilities, the orientational order parameter and average angle between the longitudinal axis of the lipid molecule and the membrane normal, and the molecular shape factors of the lipid molecules. The values thereby obtained are in excellent agreement with parameters determined by other techniques, and provide a powerful tool for analyzing lipid-protein, protein-protein, and protein-ligand interactions in proteolipid films.

Probing the primary event in the photocycle of photoactive yellow protein using photochemical hole-burning technique.

Photochemical hole-burning spectroscopy was used to study the excited-state electronic structure of the 4-hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans-to-cis isomerization process on a femtosecond-to-picosecond time scale, similar to membrane-bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole-burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm-1 could be calculated from the deconvolution of the hole-burned spectra leading to an estimated dephasing time of approximately 14 fs for the PYP excited-state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm-1 in the ground state to approximately 1429 cm-1 upon photoexcitation. The results obtained allowed us to speculate about the excited-state structure of PYP. We discuss the data for PYP in relation to the excited-state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck-Condon envelope. The origin of this spectrum was tentatively assigned.

Highly nonproductive complexes with Anabaena ferredoxin at low ionic strength are induced by nonconservative amino acid substitutions at Glu139 in Anabaena ferredoxin:NADP+ reductase.

Ferredoxin (Fd) and ferredoxin:NADP(+) reductase (FNR) from Anabaena function in photosynthetic electron transfer (et). The et interaction between the FNR charge-reversal mutant E139K and Fd at 12 mM ionic strength (mu) is extremely impaired relative to the reaction with wt FNR, and the dependency of k(obs) on E139K concentration shows strong upward curvature at protein concentrations > or = 10 microM. However, at values of mu > or = 200 mM, reaction rates approach those of wild-type FNR, and normal saturation kinetics are observed. For the E139Q mutant, which is also significantly impaired in its et interaction with Fd at low FNR concentrations and low mu values, the dependency of k(obs) on E139Q concentration shows a smaller degree of upward curvature at mu = 12 and 100 mM and shows saturation kinetics at higher values of mu. wt FNR and the E139D mutant both show a slight amount of upward curvature at FNR concentrations >30 microM at mu = 12 mM but show the expected saturation kinetics at higher values of mu. These results are explained by a mechanism in which the mutual orientation of the proteins in the complex formed at low ionic strength with the E139K mutant is so far from optimal that it is almost unreactive. At increased E139K concentrations, the added mutant FNR reacts via a collisional interaction with the reduced Fd present in the unreactive complex. The et reactivity of the low ionic strength complexes depends on the particular amino acid substitution, which via electrostatic interactions alters the specific geometry of the interface between the two proteins. The presence of a negative charge at position 139 of FNR allows the most optimal orientations for et at ionic strengths below 200 mM.

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein.

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.

Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy.

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.

Plasmon resonance studies of agonist/antagonist binding to the human delta-opioid receptor: new structural insights into receptor-ligand interactions.

Structural changes accompanying the binding of ligands to the cloned human delta-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.

Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies.

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.

The FMN to heme electron transfer in cytochrome P450BM-3. Effect of chemical modification of cysteines engineered at the FMN-heme domain interaction site.

The crystal structure of the complex between the heme and FMN-containing domains of Bacillus megaterium cytochrome P450BM-3 (Sevrioukova, I. F., Li, H., Zhang, H., Peterson, J. A., and Poulos, T. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1863-1868) indicates that the proximal side of the heme domain molecule is the docking site for the FMN domain and that the Pro(382)-Gln(387) peptide may provide an electron transfer (ET) path from the FMN to the heme iron. In order to evaluate whether ET complexes formed in solution by the heme and FMN domains are structurally relevant to that seen in the crystal structure, we utilized site-directed mutagenesis to introduce Cys residues at positions 104 and 387, which are sites of close contact between the domains in the crystal structure and at position 372 as a control. Cys residues were modified with a bulky sulfhydryl reagent, 1-dimethylaminonaphthalene-5-sulfonate-L-cystine (dansylcystine (DC)), to prevent the FMN domain from binding at the site seen in the crystal structure. The DC modification of Cys(372) and Cys(387) resulted in a 2-fold decrease in the rates of interdomain ET in the reconstituted system consisting of the separate heme and FMN domains and had no effect on heme reduction in the intact heme/FMN-binding fragment of P450BM-3. DC modification of Cys(104) caused a 10-20-fold decrease in the interdomain ET reaction rate in both the reconstituted system and the intact heme/FMN domain. This indicates that the proximal side of the heme domain molecule represents the FMN domain binding site in both the crystallized and solution complexes, with the area around residue 104 being the most critical for the redox partner docking.

Dual photoactive species in Glu46Asp and Glu46Ala mutants of photoactive yellow protein: a pH-driven color transition.

Photoactive yellow protein (PYP) is a blue light sensor present in the purple photosynthetic bacterium Ectothiorhodospira halophila, which undergoes a cyclic series of absorbance changes upon illumination at its lambda(max) of 446 nm. The anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen-bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becomes protonated in the photobleached state (I(2)) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent. In previous mutagenesis studies of a Glu46Gln mutant, we have shown that a key factor in controlling the color and photocycle kinetics of PYP is this H-bonding system. To further investigate this, we have now characterized Glu46Asp and Glu46Ala mutants. The ground-state absorption spectrum of the Glu46Asp mutant shows a pH-dependent equilibrium (pK = 8.6) between two species: a protonated (acidic) form (lambda(max) = 345 nm), and a slightly blue-shifted deprotonated (basic) form (lambda(max) = 444 nm). Both of these species are photoactive. A similar transition was also observed for the Glu46Ala mutant (pK = 7.9), resulting in two photoactive red-shifted forms: a basic species (lambda(max) = 465 nm) and a protonated species (lambda(max) = 365 nm). We attribute these spectral transitions to protonation/deprotonation of the phenolate oxygen of the chromophore. This is demonstrated by FT Raman spectra. Dark recovery kinetics (return to the unphotolyzed state) were found to vary appreciably between these various photoactive species. These spectral and kinetic properties indicate that the hydrogen bond between Glu46 and the chromophore hydroxyl group is a dominant factor in controlling the pK values of the chromophore and the glutamate carboxyl.

Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NADP+ reductase: site-specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials.

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein-protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein-protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.

The pH dependence of intramolecular electron transfer rates in sulfite oxidase at high and low anion concentrations.

The individual rate constants for intramolecular electron transfer (IET) between the Mo(VI)Fe(II) and Mo(V)Fe(III) forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO4(2-) and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from Fe(II) to Mo(VI) decreases with increasing pH, both in the presence and absence of 50 mM SO4(2-) . However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO4(2-), and increases with pH in the presence of 50 mM S04(2-). This behavior is consistent with a mechanism in which IET from Mo(V) to Fe(III) is coupled to proton transfer from Mo(V)-OH to OH-, and the reverse IET process is coupled to proton transfer from H2O to Mo(VI) = O. At high concentrations of small anions, direct access of H2O or OH- to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton when an anion blocks direct access of H2O or OH- to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.