Total Absorbance Change Research Articles

Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4

We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s(-1). Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR-CYP3A4 complex can dissociate and reform between the first and second electron transfers.

Clearing of Suspensions of Micrococcus lysodeikticus Catalysed by Lysozymes from Hen, Goose, and Turkey Egg Whites, Human Milk, and Phage T4. Assessment of Potential as Signal Generators for Homogeneous Enzyme Immunoassays for Urinary Steroids

Lysozymes (3.2.1.17) from goose (Anser anser) egg white, turkey (Melagris gallopavo) egg white, phage T4 and human milk were compared with hen egg white lysozyme in their ability to clear a suspension of Micrococcus lysodeikticus. All of the lysozymes, except hen egg white lysozyme, catalysed the clearing of the Micrococcus lysodeikticus suspension in a biphasic fashion. Compared to hen egg white lysozyme, the total absorbance or transmission change over 5 and 20 minutes was less in all cases, except for human lysozyme. Human lysozyme was, therefore, a potential alternative, more rapid signal generator for the measurement of urinary estrone glucuronide excretion rates because of its structural similarity to hen egg white lysozyme. The apparent KM values for hen egg white lysozyme increased with the enzyme concentration.

Evidence of acid–base interactions between amines and model indoor surfaces by ATR-FTIR spectroscopy

Molecular associations of pyridine with cellulose and gypsum, surrogates for common indoor surface materials, were studied using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectrophotometric method. The purpose of this study was to gain insight into the molecular interactions of amines with well-characterized materials that affect their partitioning between indoor air and surfaces. The experimental results suggest the presence of at least two sorptive states for volatile and semivolatile amines, attributed to the chemisorbed species and to a more labile surface state (i.e., physisorbed pyridine). Both exhibited spectroscopic signatures corresponding to aromatic C–H stretching modes (2950–3100 cm −1) in the studied spectral region. Chemisorbed pyridine could be identified by the presence of additional IR signals in the N–H and O–H stretching region of the spectrum (2900–3600 cm −1). During desorption under a stream of N 2, surface enrichment in the chemisorbed species was evidenced by a slower reduction of the absorbance of the broad band at 2900–3600 cm −1 in relation to the total pyridine absorbance change. This spectroscopic evidence for acid–base interactions between amines and surfaces is consistent with the desorption behavior observed in previous work for nicotine from model surfaces.

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.

Kinetic equivalence of the subunits of liver alcohol dehydrogenase.

1 The kinetics of two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism have been analyzed theoretically with respect to the precise magnitude of amplitudes of the two slowest transients governing such reactions under single-turnover conditions. Relationships derived have been applied to the liver alcohol dehydrogenase system for detailed interpretation of amplitudes of the biphasic absorbance changes associated with the enzymic reduction of aromatic aldehydes in catalytic-suicide experiments. 2 Amplitudes of the slow phase 328–330-nm absorbance changes occurring during the enzymic reduction of various para-substituted benzaldehydes by NADH or NAD2H correspond to 6–26% of the total absorbance change under conditions of substrate and coenzyme saturation. Saturation values of amplitudes determined in the present and previous investigations agree within 2 % of the total absorbance change with those calculated theoretically for an ordered mechanism of enzyme action at equivalent sites. In particular, amplitudes observed exhibit the expected sensitivity to electronic substituent and deuterium isotope effects. 3 Enzyme recycling, aldehyde binding, and alcohol desorption provide negligible contributions to the 328–330-nm absorbance changes observed during aldehyde reduction in the presence of 20 mM pyrazole. The maximum slow phase amplitude contribution provided by pyrazole binding corresponds to 5–6% of the total absorbance change. Additional slow phase absorbance changes persisting at saturating concentrations of coenzyme and substrate reflect incomplete accumulation of the enzymic intermediate formed through hydride transfer in the rapid reaction phase. This intermediate contains no detectable amounts (less than 2 %,) of NADH, but exhibits spectral properties indistinguishable from those of enzyme. NAD+. alcohol complexes examined. 4 It has been concluded in recent publications that an ordered ternary-complex mechanism involving equivalent sites is insufficient to account for the transient-state kinetics of liver alcohol dehydrogenase catalysis. These conclusions are shown to be based on an inadequate interpretation of the observations made. Results reported up to date provide no evidence whatsoever for the kinetic significance of subunit interactions leading to a non-equivalence of the enzymic sites. Subunit interactions resulting in a “half-site7rdquo; reactivity of the enzyme are definitely not at hand.

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Cytochrome b(5) (cyt b(5)) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane alpha-helix at its carboxyl terminus. These two domains are connected by an approximately 15-amino acid linker domain, Ser(90)-Asp(104), which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b(5) to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b(5) to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b(5) except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b(5) to interact with cyt P450 2B4. A model for the membrane-bound cyt b(5)-cyt P450 complex is presented.

FTIR investigation of polymerisation and polyacid neutralisation kinetics in resin-modified glass-ionomer dental cements

A new diamond ATR FTIR method has been developed to quantify the processes occurring in the resin-modified glass-ionomer cement (RMGIC), Fuji II LC (Improved), at 1 mm depth from the cement/water interface. With Fuji II LC (Improved) various changes in the spectra due to 90% monomer polymerisation were observed within 1 min after 20 s exposure to a dental light. Following polymerisation further different peak shifts with time were detected. Comparison with spectral changes seen during setting of the conventional glass-ionomer cement, Fuji IX, showed these could be assigned to water sorption and/or polyacid neutralisation. Any absorbance change due to the acid/glass reaction alone exhibited 2 linear regions when plotted against square root of time. Such behaviour suggests two separate diffusion mechanisms for acid neutralisation. The first faster one ceases at 30 or 150 min after mixing in Fuji IX and Fuji II LC (Improved), respectively. It was proposed that these were the times at which all the water (a required component of the reaction) in the original formulation is used up. The slower process was the same acid/glass reaction but initiated by water sorption. The initial rates of absorbance change due to acid neutralisation were 17 times faster for Fuji IX than Fuji II LC (Improved). By 4 days however, the total absorbance change due to acid neutralisation for Fuji IX was only 4 times that for Fuji II LC (Improved). Such results can help to explain changes in cement properties with time.

Simultaneous Determination of Micro Bromide and Iodide by Kinetic Spectrophotometric Method

ABSTRACT ABSTRACTA new kinetic spectrophotometric method for the simultaneous determination of concentrations of micro bromide and iodide has been proposed. It is based on their catalytic effects on the reaction of m-cresol purple oxidized by potassium periodate in hydrochloride acid medium. The reaction rate was monitored by measuring the decrease in absorbance at 528nm and the increase in absorbance at 455nm. The total difference in absorbance of the sum of bromide and iodide is identical with determination of bromide was carried out after Cr(VI) oxidized I− to I2, and I2 was removed by extraction with CCI4, and the amount of iodide was measured by subtracting the absorbance change of bromide from the total absorbance change in the presence of bromine and iodide. The optimum conditions influencing the reaction rate were studied. The linear range of determination is 0∼4.0μg/ml for Br− and 0∼3.0 μg/ml for I−. The detection limits are 0.032μg/ml for Br− and 0.059 μ/ml for I−. The method was successfully applied to the determination of micro amounts of bromide and iodide in food and life samples.

Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli.

Cytochrome c552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem-containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid-point potential redox centres. The best fit to the experimental data is for three n=1 components with mid-point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf deletion mutant to Nrf+ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.

Synthesis and interconversion of reduced folylpolyglutamates

Tetrahydrofolate (THF) is the coenzyme for several enzymes involved in one-carbon metabolism. The physiological form of THF contains a chain of glutamate residues linked through the γ-carboxyl group. To accommodate the naming of these derivatives, they are named as polyglutamate derivatives of tetrahydropteroate (HaPte), which has no glutamate residue. Tetrahydrofolate then becomes HaPteGlu and the pentaglutamate form HaPteGlu 5 . This chapter describes a method for synthesizing 5-formyl-H 4 PteGlu n , where n is 1 to 6, and the interconversion of this derivative to the other one-carbon derivatives. The concentration of H 4 PteGlu n is determined by measuring the increase in absorbance at 340 nm following the addition of 20 μ g of C 1 -THF synthase to a solution of HaPteGlu n , 0.1 m M NADP + , 20 mi L -serine, 0.2 mg of serine hydroxymethyltransferase (SHMT) in 1 ml of potassium N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonate (KBES), pH 7.3, containing 5 m M 2-mercaptoethanol. The HnPteGlu n concentration is obtained by dividing the total absorbance change at 340 nm by the extinction coefficient of 7200 M –1 cm –1 , which is the result of the combined absorbance at 340 nm of NADPH and 5,10-methenyltetrahydrofolate at pH 7.3. This extinction coefficient of 7200 M -1 cm -1 changes with pH, so it is important that the pH be pH 7.3.

A Metabolic Enzyme That Rapidly Produces Superoxide, Fumarate Reductase of Escherichia coli

Aerobic organisms synthesize superoxide dismutases in order to escape injury from endogenous superoxide. An earlier study of Escherichia coli indicated that intracellular superoxide is formed primarily by autoxidation of components of the respiratory chain. In order to identify those components, inverted respiratory vesicles were incubated with five respiratory substrates. In most cases, essentially all of the superoxide was formed through autoxidation of fumarate reductase, despite the paucity of this anaerobic terminal oxidase in the aerobic cells from which the vesicles were prepared. In contrast, most dehydrogenases, the respiratory quinones, and the cytochrome oxidases did not produce any detectable superoxide. The propensity of fumarate reductase to generate superoxide could conceivably deluge cells with superoxide when anaerobic cells, which contain abundant fumarate reductase, enter an aerobic habitat. In fact, deletion or overexpression of the frd structural genes improved and retarded, respectively, the outgrowth of superoxide dismutase-attenuated cells when they were abruptly aerated, suggesting that fumarate reductase is a major source of superoxide in vivo. Steric inhibitors that bind adjacent to the flavin completely blocked superoxide production, indicating that the flavin, rather than an iron-sulfur cluster, is the direct electron donor to oxygen. Since the turnover numbers for superoxide formation by other flavoenzymes are orders of magnitude lower than that of fumarate reductase (1600 min-1), additional steric or electronic factors must accelerate its autoxidation.

Rapid refolding of native epitopes on the surface of cytochrome c.

Refolding of surface epitopes on horse cytochrome c has been measured by monoclonal antibody binding. Two antibodies were used to probe re-formation of native-like surface structure: one antibody (2B5) binds to native cytochrome c near a type II turn (residue 44) while the other (5F8) binds to a different epitope on the opposite face of the protein near the amino terminus of an alpha-helical segment (residue 60). The results show that within the first approximately 100 ms of refolding all of the unfolded protein collapses to native-like folding intermediates that contain both antibody binding sites. All three absorbance/fluorescence-detected kinetic phases in the folding of cytochrome c (k1 approximately 5 s-1, k2 approximately 0.4 s-1, k3 approximately 0.03 s-1) are slower than the rates of re-formation of the antibody binding sites (k(obs) > 10.0 s-1), suggesting that the formation of antibody binding sites precedes the refolding reactions observed in kinetically resolved optically-detected refolding phases. Kinetically unresolved folding processes account for 79% and 19% of the total fluorescence change and absorbance change, respectively, observed in equilibrium unfolding. Thus, kinetically unresolved folding reactions appear to be responsible for re-formation of the MAb binding sites within partially folded intermediate species. These species are non-native (incompletely folded) in that their optical properties are in between those of the unfolded and the fully folded protein. As a test of whether antibody binding to folding intermediate(s) perturbs further folding, the rate of the absorbance-detected slow refolding phase has been measured for folding intermediate(s) of cytochrome c complexed with antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of thermal lens spectrometry to kinetic speciation studies of metal ions in natural water models with colloidal ligands

Thermal lensing spectrometry (TLS) has been applied to measurement of kinetics with the aim of extending kinetic speciation methods to the low concentrations of metal ions in the presence of colloidal ligands which are important in many natural systems. Using bromination of acetone as a test reaction, it is shown thatn 2–3 significant figure rate constants can be obtained from runs with a total absorbance change of < 0.003. Results are presented on TLS kinetic analysis of hydrous ferric oxide colloids hydrolyzing in the presence of sulfosalicylic acid (SSA) to give FeSSA and on the kinetic analysis of Cu(II) bound to hydrous ferric oxides. In both cases, it is seen that lability is increased at the lower concentrations accessed by TLS as compared to behavior previously reported at concentrations required for analysis by conventional spectrophotometry. That this is a change of the colloids and not a disagreement between TLS and conventional methods is confirmed by demonstrating agreement between the two methods at different concentrations of samples of Cu(II) ion exchanged into zeolite particles of defined size. Noise levels were higher than in bromination of acetone. Studies using colloidal latex particles of known size show that light scattering by nonabsorbing particles causes no interference in TLS absorbance measurements but homodisperse hematite colloids which absorb at the pump laser wavelength cause a significant noise increase.

Flow-flash study of the reaction between cytochrome bo and oxygen

The reaction between reduced cytochrome bo from Escherichia coli and oxygen has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. Absorbance changes were monitored in the alpha and Soret spectral regions. Two kinetic phases taking place at catalytically competent rates could be detected. The apparent rate constant obtained for both the first and second phase showed a hyperbolic dependence on the oxygen concentration. For the first phase, we obtained limiting first- and second-order rate constants at saturating and low oxygen concentrations of 4.5 x 10(4) s-1 and 1.6 x 10(8) M-1 s-1, respectively. The corresponding values for the second phase were 5 x 10(3) s-1 and 1.7 x 10(7) M-1 s-1. The first phase accounted for 30% of the total absorbance change in the Soret band (430 nm) and 15% of the total absorbance change in the alpha band (555 nm). These reactions are followed by a very slow phase with a lifetime of about 1 s. We have also studied the interaction between the fully oxidized enzyme and hydrogen peroxide, and we have found that peroxide binding induces an absorbance increase in the alpha band and a red shift of the Soret band. A consideration of the magnitude of the absorbance changes taking place during the first phase suggests that this reaction includes at least partial oxidation of the low-spin cytochrome b.

Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum

Here we describe an error-compensating kinetic-based method for the enzymatic quantification of creatinine in serum. The method, which has a large linear range and very low dependency on experimental variables that influence enzyme activity, is based on the use of creatinine amidohydrolase in a four-step coupled reaction sequence to generate a product that is monitored photometrically. We collected data for absorbance vs time during two to four half-lives of each reaction and fit a first-order model to the data to compute the total absorbance change that would be measured if the reaction were monitored to completion. Computed values of absorbance change agreed well with measured values and varied linearly with creatinine concentration in the sample throughout the range examined: 44 to 1326 mumol/L. A twofold change in enzyme activity in the final reaction mixture (3.3 to 6.6 kU/L) produced changes of only 12% and 4% for creatinine concentrations of 44 and 353 mumol/L, respectively. Results (y) for 39 serum samples that contained creatinine concentrations between 20 and 1800 mumol/L agreed well with liquid-chromatographic results (x), yielding linear least-squares statistics of y = (1.03 +/- 0.01) x + (5 +/- 5) mumol/L (r = 0.995, Sy.x = 37 mumol/L). We conclude that the predictive kinetic approach is a robust method for the quantification of creatinine in serum.

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.

Cysteine Residues in the Active Site of Corynebacterium Sarcosine Oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 is inhibited by iodoacetamide (IAM) and the inhibition is prevented by the substrate analog, sodium acetate. To elucidate the mechanism of inhibition of the enzyme by IAM, we determined the amino acid sequences around the IAM-reactive cysteine residues, and the effects of the modification on the enzyme activity and the oxidation-reduction of the FAD moieties of the enzyme. The enzyme was specifically labeled with [14C]IAM, and the labeled subunit B was digested with trypsin and chymotrypsin. The HPLC profiles of the proteolytic digests showed mainly two radioactive peaks. The 14C-labeled peptides were purified, and their N-terminal sequences were determined to be Cys-Gly-Thr-Pro-Gly-Ala-Gly-Tyr (TC-1) and Ala-Gly-Ile-Ala-Cys-Xaa-Asp-Xaa-Val-Ala(-)- (TC-2). Peptide TC-2 contains a covalent FAD-binding sequence [Asx-His-Val-Ala; Shiga et al. (1983) Biochem. Int., 6, 737]. [14C]IAM-incorporation into the TC-1 sequence was strongly inhibited by sodium acetate. The N-terminal amino acid sequence of the CNBr fragment containing the TC-1 sequence (65 residues) was determined. According to the secondary structure predictions, Gly-Thr-Pro-Gly-Ala-Gly of the TC-1 sequence is located between the beta sheet and alpha helix of the sequence, indicating the presence of an AMP-binding site in the TC-1 region. The activity of the enzyme treated with IAM in the presence and absence of sodium acetate was not inhibited by sodium sulfite, which is known to react specifically with covalent FAD.(ABSTRACT TRUNCATED AT 250 WORDS)

Oscillating enzyme-bound NADH in glycolysis

In glycolyzing cell-free cytoplasmic medium extracted from yeast Saccharomyces cerevisiae, the action spectrum of oscillation has an absorption maximum around 335 nm, nearly coinciding with that of the yeast alcohol dehydrogenase (ADH)-NADH complex due to its bound NADH. Our approximate calculations based on the amount of this enzyme and coenzyme NADH present in the extract suggest that the ADH-NADH complex alone can account for 90% or more of the total absorbance change.

Conformational change accompanies redox reactions of the tetraheme cytochrome c-554 of Nitrosomonas europaea.

Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential. We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H2O2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength. Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to ≈300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.