Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.

Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.     

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.     

C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH

Glutamate decarboxylase (GAD) from Lactobacillus brevis is a very promising candidate for biosynthesis of GABA and various other bulk chemicals that can be derived from GABA. However, no structure of GAD of this origin has been reported to date, which limits enzyme engineering strategy to improve its properties for better use in production of GABA. Bacterial GAD exhibits an acidic pH optimum and there is often a sharp pH dependence. In the present work, site-directed mutagenesis was performed to delete the C-terminal residues of GAD to generate a mutant, designated as GADΔC, which exhibited extended activity toward near-neutral pH compared to the wild type. Comparison of the UV-visible, fluorescence and Circular Dichroism spectra of the mutant with those of the wild type revealed that the microenvironment of the active site had been changed. Based on the homology model, we speculated that the substrate entrance was probably enlarged in GADΔC. These results provide evidence for the important role of C-terminal region in the pH-dependent regulation of enzyme activity, and the resulting mutant would be useful in a bioreactor for continuous production of GABA.     

Application of photoactivatable fluorescent active-site directed probes to serine-containing enzymes

A photoactivatable fluorescent anthraniloyl group has been directed to the active-site serine group of alpha-chymotrypsin and trypsin. The acylated derivatives are nonfluorescent until irradiated. When activated by light a highly reactive nitrene is generated which is capable of covalent insertion into the protein matrix. The resultant insertion product of this photolysis is a highly fluorescent reporter group which has little rotational mobility and is cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked through the serine to the protein matrix in the active site region of the protein. Because of the sensitivity to the polarity of the environment shown by the anthraniloyl chromophore, the dipolar relaxation characteristics of the cross-linked enzyme and deacylated enzyme were determined. These measurements show that little relaxation occurs on the nanosecond time scale for the cross-linked enzyme, but upon deacylation of the serine increased dipolar relaxation of the protein with the attached reporter group is observed. The use of these active-site directed photoactivatable fluorescent probes can be extended to probe the active-site structure of complex enzymes and conformational dynamics of active-site regions in proteins and to serve as potential functional site labels in fluorescence resonance energy transfer measurements.     

On the nanosecond mobility in proteins: Edge excitation fluorescence red shift of protein-bound 2-(p-toluidinylnaphthalene)-6-sulfonate

The fluorescence spectra of 2-(p-toluidinylnaphthalene)-6-sulfonate associated with β-lactoglobulin, β-casein. and bovine and human serum albumins are shown to depend on excitation wavelength. A long-wave shift of the spectra is observed at the long-wave edge excitation, reaching 10 nm and above. A similar phenomenon is found in glucose glass and in glycerol at + 1°C, i.e., in systems with delayed dipolar solvent relaxation, but not in liquid solutions. This phenomenon is proposed to be based on relaxation processes in the excited state. There exists a distribution of chromophore microstates with different interactions with surrounding groups which results in heterogeneous broadening of the electronic spectra and allows photoselection of a part of this distribution, being characterized by a low transition energy. The fast structural relaxation results in an altered distribution and, if this is the case, the effect of edge excitation of fluorescence spectra is not observed. If the structural relaxation during the excited state lifetime is absent, this effect is maximal. This interpretation is in agreement with results on the influence of red edge excitation on the low-temperature fluorescence spectra of dyes and with the data on time-resolved nanosecond fluorescence spectroscopy. The results of this work strongly support the significant dye fluorescence spectral shifts on protein binding, being determined not only by polarity changes in their environment, but also by relaxation properties of protein groups in this environment. These results also indicate that on the nanosecond time scale, the structural relaxation around the excited chromophore in proteins may be incomplete.     

A mutant sarcosine oxidase in which activity depends on flavin adenine dinucleotide

The covalent flavin attachment site in the Arthrobacter sarcosine oxidase (cysteine at position 318) was replaced with serine, and the mutational effect of C318S was analyzed. Wild type and C318S with a C-terminal 6-histidine tag were constructed and homogeneously purified by the single step. The covalently binding to flavin was not essential to the enzyme activity because the C318S mutant exhibited extremely weak activity. Moreover, the activity of the mutant was recovered in the presence of flavin adenine dinucleotide (FAD), and significantly increased as the concentration of FAD increased. This dependence of the mutant on FAD indicates that the noncovalent binding of free FAD to the mutant enzyme is reversible.     

Nanosecond dynamics of charged fluorescent probes at the polar interface of a membrane phospholipid bilayer

Molecular relaxation fluorescence methods were applied to analyze the nature and characteristic times of motions of amphiphilic molecules absorbed in the polar region of a phospholipid bilayer. The fluorescence probes 2-toluidinonaphthalene-6-sulfonate and 1-anilinonaphthalene-8-sulfonate in egg phosphatidylcholine vesicles were studied. The methods of edge excitation fluorescence red shifts, nanosecond time-resolved spectroscopy, fluorescence quenching by hydrophilic and hydrophobic quenchers and emission wavelength dependence of polarization were used. The structural (dipolar) relaxation is shown to be a very rapid (subnanosecond) process. The observed nanosecond phenomena are related to translational movement of the chromophore itself towards a more polar environment and its rotation. The polar surface area of the phospholipid membrane appears to be a highly mobile liquid-like system.     

Nanosecond dynamics of a mimicked membrane-water interface observed by time-resolved stokes shift of LAURDAN

We studied the dipolar relaxation of the surfactant-water interface in reverse micelles of AOT-water in isooctane in the nanosecond and subnanosecond time ranges by incorporating the amphipathic solvatochromic fluorescent probes LAURDAN and TOE. A negative component was observed in the fluorescence decays in the red edge of the emission spectrum-the signature of an excited state reaction-with LAURDAN but not for TOE. The deconvolution of the transient reconstructed spectra of LAURDAN based on a model constructed by adding together three log-normal Gaussian equations made it possible to separate the specific dynamic solvent response from the intramolecular excited state reactions of the probe. The deconvoluted spectrum of lowest energy displayed the largest Stokes shift. This spectral shift was described by unimodal kinetics on the nanosecond timescale, whereas the relaxation kinetics of water-soluble probes have been reported to be biphasic (on the subnanosecond and nanosecond timescales) due to the heterogeneous distribution of these probes in the water pool. Most of this spectral shift probably resulted from water relaxation as it was highly sensitive to the water to surfactant molar ratio (w(0)) (60-65 nm at w(0) = 20-30). A small part of this spectral shift (9 nm at w(0) = 0) probably resulted from dipolar interaction with the AOT polar headgroup. The measured relaxation time values were in the range of the rotational motion of the AOT polar headgroup region as assessed by LAURDAN and TOE fluorescence anisotropy decays.     

Steady-state and time-resolved fluorescence studies on wild type and mutant chromatium vinosum high potential iron proteins: holo- and apo-forms

Detailed circular dichroism (CD), steady-state and time-resolved tryptophan fluorescence studies on the holo- and apo- forms of high potential iron protein (HiPIP) from Chromatium vinosum and its mutant protein have been carried out to investigate conformational properties of the protein. CD studies showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster does not have any effect on formation of secondary structure in the protein. Steady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur ([Fe(4)S(4)](3+)) cluster from the protein leads to an increase in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. CD studies on the holo- and apo- HiPIP also showed that removal of the metal prosthetic group drastically affects the tertiary structure of the protein. Time-resolved fluorescence decay of the wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fluorescence decay was also analyzed by maximum entropy method (MEM). The results of the MEM analysis agreed with those obtained from discrete exponentials model analysis. Studies on the wild type and mutants helped to assign the fast picosecond lifetime component to the W80 residue, which exhibits fast fluorescence energy transfer to the [Fe(4)S(4)](3+) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calculated from the time-resolved fluorescence results at different emission wavelengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solvent.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

黄化油菜突变体Cr3529子叶类囊体膜光谱性质研究

以发育10d的黄化油菜突变体为材料,分析了突变体油菜子叶类囊体膜的色素含量、室温吸收光谱、叶绿素荧光发射和激发光谱以及蛋白内源荧光光谱的变化。数据显示：与野生型相比,突变体油菜子叶类囊体膜的光合色素Chl α和Chl b含量均减少．但Chl α／b比值升高;突变体油菜子叶类囊体膜叶绿素捕光能力和受激发能力均下降,且较依赖于Chl α捕光并将光能激发传递给PSⅡ反应中心;突变体油菜子叶类囊体膜的蛋白内源荧光也明显异于野生型。进一步表明突变体油菜子叶类囊体膜蛋白组成发生了改变。     

Deletion mutagenesis of human cystathionine beta-synthase. Impact on activity,oligomeric status,and S-adenosylmethionine regulation

Cystathionine beta-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5'-phosphate-dependent condensation of serine and homocysteine to cystathionine. We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme. Western blot analysis of these mutants expressed in Escherichia coli indicated that residues 497-543 are involved in tetramer formation. Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity. Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme. Expression of this double-deletion mutant as a glutathione S-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner. The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme. The C-terminal region of human cystathionine beta-synthase contains two hydrophobic motifs designated "CBS domains." Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability. Removal of both of these domains resulted in stable constitutively activated enzyme. Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation by S-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.     

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant K_A = 7.0(± 0.8) × 10⁴ M^− 1. Taken together, the FAD–protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.     

The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP⁺ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.     

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.     

Mechanism of flavin reduction in the class 1A dihydroorotate dehydrogenase from Lactococcus lactis

Dihydroorotate dehydrogenases (DHODs) oxidize dihydroorotate (DHO) to orotate (OA) using the FMN prosthetic group to abstract a hydride equivalent from C6 and a protein residue (cysteine for class 1A DHODs) to deprotonate C5. The fundamental question of whether the scission of the two DHO C-H bonds is concerted or stepwise was addressed for the class 1A enzyme from Lactococcus lactis by determining kinetic isotope effects (KIEs) on flavin reduction in anaerobic stopped-flow experiments. Isotope effects were determined at two pH values. At pH 7.0, KIEs were approximately 2-fold for DHO labeled singly at the 5-position or the 6-position and approximately 4-fold for DHO labeled at both the 5- and 6-positions. At pH 8.5, the KIEs observed for DHO labeled at the 5-position, the 6-position, and the 5- and 6-positions were approximately 2-, approximately 3-, and approximately 6-fold, respectively. These isotope effects are consistent with a concerted oxidation of DHO. The pH dependence of reduction was also determined, and a pKa of 8.3 was found. This pKa can be attributed to the ionization of the active site cysteine which deprotonates C5 of DHO during the reaction. To further investigate the importance of the active site base, two site-directed mutants were also studied: Cys130Ala (removal of the active site base) and Cys130Ser (replacement with the active site base used by class 2 DHODs). Both mutant enzymes exhibited binding affinities for DHO similar to that of the wild-type enzyme. Reduction of both mutants was extremely slow compared to that of the wild type; the rate of reduction increased with pH, showing no sign of a plateau. Interestingly, double-deuterium isotope effects on the Cys130Ser mutant also showed a concerted mechanism for flavin reduction.     

Characteristics and Crystallization of Nitrogenase MoFe Protein from a nif Z Deleted Strain of Azotobacter vinelandii

△nifZ MoFe protein purified from a nifZ deleted strain of Azotobacter vinelandii (DJ194) was shown to be pure by SDS-Polyacrylamide gel electrophoresis. The protein contained 1.5 Mo atoms and 15.9 Fe atoms per molecule, the ratio of Fe to Mo was lower than that of the MoFe protein purified from the wild type strain of A. vinelandii; and Call2, H+ -reduction activity and their ratio (C2H4/H2 (Ar)) were 16.6%, 21.7% and 77.2% of those of the wild type MoFe protein, respectively. Under a somewhat different condition from that for the crystallization of the wild type MoFe protein dark brown rhombohedron crystals of △nifZ MoFe protein were obtained. It indicated that the deletion of the △nif Z resulted in the decrease of number or change in the structure of P-cluster in the mutant MoFe protein, which caused the significant structured and function of change of the protein.     

Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

Red-edge-excitation fluorescence spectroscopy of indole and tryptophan

Studies on the dependence of indole and tryptophan fluorescence emission spectra on excitation wavelength, ^ex, show that the emission shifts to longer wavelengths for red-edge excitation in different solid and viscous solvents. In solid systems the spectral shifts for excitation in the range from 290 to 310 nm can reach tens of nm, and they are more significant than changes of ^ex. In a viscous medium the magnitude of this effect is shown to be directly related to the dipole-reorientational relaxation of solvent molecules in the environment of the chromophore, which allows the relaxation times to be estimated. The method involves simple steady-state measurements of fluorescence spectra at the maximum and at the red edge of the absorption band. Since it is not necessary to obtain information on the fluorescence spectra of completely relaxed states, this method for the estimation of relaxation times may have advantages in studies of proteins compared with the conventional relaxation shift method, and may produce complementary information to that obtained by nanosecond time-resolved spectroscopy.     

Yeast dihydroxybutanone phosphate synthase,an enzyme of the riboflavin biosynthetic pathway,has a second unrelated function in expression of mitochondrial respiration

aE280/U1 is a pet mutant of Saccharomyces cerevisiae partially deficient in cytochromes a, a3, and cytochrome b. The ability of this mutant to respire is restored by RIB3, a gene previously shown to code for 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBP synthase), an enzyme of the riboflavin biosynthetic pathway. The sequences of RIB3 from wild type and aE280/U1 indicated a single base change resulting in an A137T substitution. The alanine 137 is a conserved residue located in a cavity on the surface of the protein distant from the active site and from the subunit interaction domain involved in homodimer formation. The respiratory defect elicited by this mutation cannot be explained by a flavin insufficiency based on the following evidence: 1) growth of the aE280/U1 on respiratory substrates is not rescued by exogenous riboflavin; 2) the levels of flavin nucleotides are not significantly different in the mutant and wild type. We proposed that in addition to its known function in riboflavin synthesis, RIB3 also functions in expression of mitochondrial respiration. Restoration by riboflavin of growth of a rib3 deletion mutant on glucose but not glycerol/ethanol also supported this conclusion. An antibody against the N-terminal half of DHBP synthase was used to study its subcellular distribution. Most of the protein was localized in the cytosolic fraction, but a small fraction was detected in the mitochondrial intermembrane space.