Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase.

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.

Mechanism of carotenoid singlet excited state energy transfer in modified bacterial reaction centers.

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules. On the donor side, carotenoids with various extents of pi-electron conjugation were examined. RCs studied include those from the anaerobically grown wild-type strain containing the carotenoid spheroidene, which has 10 conjugated carbon-carbon double bonds; the GA strain containing neurosporene, which has nine conjugated double bonds; and aerobically grown wild-type cells, as well as aerobically grown H(M182)L mutant, both containing the carbonyl-containing carotenoid spheroidenone, which has 11 conjugated double bonds. By varying the structure of the carotenoid, we observed the effect of altering the energies of the carotenoid excited states on the rate of energy transfer. Both S(1)- and S(2)-mediated carotenoid-to-bacteriochlorophyll energy transfer processes were observed. The highest transfer efficiency, from both the S(1) and S(2) states, was observed using the carotenoid with the shortest chain. The S(1)-mediated carotenoid-to- bacteriochlorophyll energy transfer efficiencies were determined to be 96%, 84%, and 73% for neurosporene, spheroidene, and spheroidenone, respectively. The S(2)-mediated energy transfer efficiencies follow the same trend but could not be determined quantitatively because of limitations in the time resolution of the instrumentation. The dependence of the energy transfer rate on the energetics of the energy transfer acceptor was verified by performing measurements with RCs from the H(M182)L mutant. In this mutant, the bacteriochlorophyll (denoted B(B)) located between the carotenoid and the RC special pair (P) is replaced by a bacteriopheophytin (denoted phi(B)), where the Q(X) and Q(Y) bands of phi(B) are 1830 and 1290 cm(-1), respectively, higher in energy than those of B(B). These band shifts associated with phi(B) in the H(M182)L mutant significantly alter the spectral overlap between the carotenoid and phi(B), resulting in a significant decrease of the transfer efficiency from the carotenoid S(1) state to phi(B). This leaves energy transfer from the carotenoid S(2) state to phi(B) as the dominant channel. Largely because of this change in mechanism, the overall efficiency of energy transfer from the carotenoid to P decreases to less than 50% in this mutant. Because the spectral signature of phi(B) is different from that of B(A) in this mutant, we were able to demonstrate clearly that the carotenoid-to-P energy transfer is via phi(B). This finding supports the concept that, in wild-type RCs, the carotenoid-to-P energy transfer occurs through the cofactor located at the B(B) position.

Kinetics and thermodynamics of phenotype: unwinding and rewinding the nucleosome.

Chromatin "remodeling" is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA-histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be -32k(B)T for nucleosomes containing unmodified H4 and -27k(B)T for nucleosomes containing H4-R45H. Thus, the "loosening" or "remodeling" caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5k(B)T. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.

A new bright green-emitting fluorescent protein--engineered monomeric and dimeric forms.

Fluorescent proteins have become essential tools in molecular and biological applications. Here, we present a novel fluorescent protein isolated from warm water coral, Cyphastrea microphthalma. The protein, which we named vivid Verde fluorescent protein (VFP), matures readily at 37 degrees C and emits bright green light. Further characterizations revealed that VFP has a tendency to form dimers. By creating a homology model of VFP, based on the structure of the red fluorescent protein, DsRed, we were able to make mutations that alter the protein's oligomerization state. We present two proteins, mVFP and mVFP1, that are both exclusively monomeric, and one protein, dVFP, which is dimeric. We characterized the spectroscopic properties of VFP and its variants in comparison with enhanced green fluorescent protein (EGFP), a widely used variant of GFP. All the VFP variants are at least twice as bright as EGFP. Finally, we demonstrated the effectiveness of the VFP variants in both in vitro and in vivo detection applications.

Differences in a conformational equilibrium distinguish catalysis by the endothelial and neuronal nitric-oxide synthase flavoproteins.

Nitric oxide (NO) is a physiological mediator synthesized by NO synthases (NOS). Despite their structural similarity, endothelial NOS (eNOS) has a 6-fold lower NO synthesis activity and 6-16-fold lower cytochrome c reductase activity than neuronal NOS (nNOS), implying significantly different electron transfer capacities. We utilized purified reductase domain constructs of either enzyme (bovine eNOSr and rat nNOSr) to investigate the following three mechanisms that may control their electron transfer: (i) the set point and control of a two-state conformational equilibrium of their FMN subdomains; (ii) the flavin midpoint reduction potentials; and (iii) the kinetics of NOSr-NADP+ interactions. Although eNOSr and nNOSr differed in their NADP(H) interaction and flavin thermodynamics, the differences were minor and unlikely to explain their distinct electron transfer activities. In contrast, calmodulin (CaM)-free eNOSr favored the FMN-shielded (electron-accepting) conformation over the FMN-deshielded (electron-donating) conformation to a much greater extent than did CaM-free nNOSr when the bound FMN cofactor was poised in each of its three possible oxidation states. NADPH binding only stabilized the FMN-shielded conformation of nNOSr, whereas CaM shifted both enzymes toward the FMN-deshielded conformation. Analysis of cytochrome c reduction rates measured within the first catalytic turnover revealed that the rate of conformational change to the FMN-deshielded state differed between eNOSr and nNOSr and was rate-limiting for either CaM-free enzyme. We conclude that the set point and regulation of the FMN conformational equilibrium differ markedly in eNOSr and nNOSr and can explain the lower electron transfer activity of eNOSr.

Optical spectroscopic studies of light-harvesting by pigment-reconstituted peridinin-chlorophyll-proteins at cryogenic temperatures.

Low temperature, steady-state, optical spectroscopic methods were used to study the spectral features of peridinin-chlorophyll-protein (PCP) complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). Absorption spectra taken at 10 K provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment-protein interactions responsible for the positions of the Qy bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Qy and Soret transition energies. Fluorescence excitation spectra were used to compute energy transfer efficiencies of the various complexes at room temperature and these were correlated with previously reported ultrafast, time-resolved optical spectroscopic dynamics data. The results illustrate the robust nature and value of the PCP complex, which maintains a high efficiency of antenna function even in the presence of non-native chlorophyll species, as an effective tool for elucidating the molecular details of photosynthetic light-harvesting.

Femtosecond time-resolved absorption spectroscopy of main-form and high-salt peridinin-chlorophyll a-proteins at low temperatures.

Steady-state and femtosecond time-resolved optical methods have been used to compare the spectroscopic features and energy transfer dynamics of two systematically different light-harvesting complexes from the dinoflagellate Amphidinium carterae: main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a-proteins. Pigment analysis and X-ray diffraction structure determinations [Hofmann, E., Wrench, P. M., Sharples, F. P., Hiller, R. G., Welte, W., Diederichs, K. (1996) Science 272, 1788-1791; T. Schulte, F. P. Sharples, R. G. Hiller, and E. Hofmann, unpublished results] have revealed the composition and geometric arrangements of the protein-bound chromophores. The MFPCP contains eight peridinins and two chlorophyll (Chl) a, whereas the HSPCP has six peridinins and two Chl a, but both have very similar pigment orientations. Analysis of the absorption spectra has shown that the peridinins and Chls absorb at different wavelengths in the two complexes. Also, in the HSPCP complex, the Qy transitions of the Chls are split into two well-resolved bands. Quantum computations by modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) methods have revealed that charged amino acid residues within 8 A of the pigment molecules are responsible for the observed spectral shifts. Femtosecond time-resolved optical spectroscopic kinetic data from both complexes show ultrafast (<130 fs) and slower (approximately 2 ps) pathways for energy transfer from the peridinin excited singlet states to Chl. The Chl-to-Chl energy transfer rate constant for both complexes was measured and is discussed in terms of the Förster mechanism. It was found that, upon direct Chl excitation, the Chl-to-Chl energy transfer rate constant for MFPCP was a factor of 4.2 larger than for HSPCP. It is suggested that this difference arises from a combination of factors including distance between Chls, spectral overlap, and the presence of two additional peridinins in MFPCP that act as polarizable units enhancing the rate of Chl-to-Chl energy transfer. The study has revealed specific pigment-protein interactions that control the spectroscopic features and energy transfer dynamics of these light-harvesting complexes.

Femtosecond time-resolved absorption spectroscopy of astaxanthin in solution and in alpha-crustacyanin.

Steady-state absorption and femtosecond time-resolved spectroscopic studies have been carried out on astaxanthin dissolved in CS2, methanol, and acetonitrile, and in purified alpha-crustacyanin. The spectra of the S0 --> S2 and S1 --> S(n) transitions were found to be similarly dependent on solvent environment. The dynamics of the excited-state decay processes were analyzed with both single wavelength and global fitting procedures. In solution, the S1 lifetime of astaxanthin was found to be approximately 5 ps and independent of solvent. In alpha-crustacyanin, the lifetime was noticeably shorter at approximately 1.8 ps. Both fitting procedures led to the conclusion that the lifetime of the S2 state was either comparable to or shorter than the instrument response time. The data support the idea that dimerization of astaxanthin in alpha-crustacyanin is the primary molecular basis for the bathochromic shift of the S0 --> S2 and S1 --> S(n) transitions. Planarization of the astaxanthin molecule, which leads to a longer effective pi-electron conjugated chain and a lower S1 energy, accounts for the shorter tau1 in the protein.

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae.

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the Förster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.