James T. McFarland

Metal-flavin complexation. A resonance Raman investigation

Several groups have recently shown that high quality resonance Raman spectra can be obtained for flavin species in spite of their intense fluorescence. We are interested in obtaining the resonance Raman spectra of flavins in various chemical environments in order to determine whether the spectra are useful in probing the chemical interaction between flavins and protein in flavoenzymes. We have obtained the resonance Raman spectrum of a nonfluorescent Ag+ complex of FMN. Several large changes occur in the FMN resonance Raman spectrum upon Ag+ complexation; among these are changes in the 1580 cm-1 region of the FMN spectrum (assigned to nu C=N at N-5 and C-4a), the 1410 cm-1 region and the 1260 cm-1 region (associated with a vibration having some delta N-N-H character at N-3). Similar changes are observed in the same region of a Ru2+-FMN complex. Since these spectral changes occur in two metal flavin complexes with very different electronic spectra, they would seem to be due to vibrational changes induced by metal complexation at N-5 and the oxygen at C-4 of flavin rather than the details of the vibronic interactions which give rise to the resonance enhancement of the spectrum. A structure for the Ag+-FMN complex is suggested. This study has potential physiological significance, because it illustrates the possible role of resonance Raman spectroscopy as a tool for the determination of direct flavin metal interaction in dilute aqueous solution of metalloflavoproteins.

Intermediates during the fatty acyl CoA dehydrogenase catalyzed reduction of electron transfer flavoprotein (ETF) by fatty acyl CoA esters

Abstract The reaction kinetics for the reduction of ETF by saturated fatty acyl CoA esters has traditionally been studied by dye assays utilizing transfer of electrons from the reduced FAD of ETF to 2,6-Dichloroindophenol (DCI). We have found that it is preferable to use the natural fluorescence and absorbance properties of ETF itself in order to investigate both the steady state and single turnover reduction of ETF. Our investigations indicate formation of a red anionic semiquinone as an intermediate in the two electron reduction of ETF under steady state kinetic condition using butyryl CoA as substrate. Furthermore, V max E ∼- 6.0 sec −1 calculated from steady state kinetic data for formation of this semiquinone observed from the increase in 370 nm absorbance or decrease in ETF fluorescence emission at 510 nm (the dehydrogenase catalyst does not fluoresce) is much faster than V max E ∼- 1.0 sec −1 for hydroquinone formation. Even though DCI is a two electron acceptor, V max E ∼- 4.0 sec −1 for butyryl CoA is limited by the rate of formation of ETF semiquinone, which must, therefore, transfer its single electron to DCI. Investigation of the extent of ETF reduction to semiquinone (measured from fluorescence) by a substrate which transfers electrons quantitatively to the dehydrogenase catalyst indicates that the overall stoichiometry is two electrons transferred to the two FAD flavins on the dimeric ETF molecule. Furthermore, hydroquinone is not formed from disproportionation of the ETF semiquinone dimer since formation of hydroquinone is not accompanied by the fluorescence increase predicted from such disproportionation.

A laser Raman study of lysozyme denaturation

Abstract The Raman spectrum of chemically denatured lysozyme was studied. The denaturants studied included dimethyl sulfoxide, LiBr, guanidine · HCl, sodium dodecyl sulfate, and urea. Previous studies have shown that the amide I and amide III regions of the Raman spectrum are sensitive to the nature of the hydrogen bond involving the amide group. The intensity of the amide III band at 1260 cm−1 (assigned to strongly hydrogen-bonded α-helix structure) relative to the intensity of the amide III band near 1240 cm−1 (assigned to less strongly hydrogen-bonded groups) is used as a parameter for comparison with other physical parameters used to assess denaturation. The correlation between this Raman parameter and denaturation as evidenced by enzyme activity and viscosity measurements is good, leading to the conclusion that the amide III Raman spectrum is useful for assessing the degree of denaturation. The Raman spectrum clearly depends on the type of denaturant employed, suggesting that there is not one unique denatured state for lysozyme. The data, as interpreted, place constraints on the possible models for lysozyme denaturation. One of these is that the simple two-state model does not seem consistent with the observed Raman spectral changes.

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study☆

Abstract The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic. Addition of two [ 14 C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD + -pyrazole complex. However, the ability to bind NAD + -pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD + -pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD + -pyrazole binding capacity remains; under these reaction conditions one [ 14 C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD + -pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD + during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD + -pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD + -pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase. Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.

Resonance Raman studies of ETF dehydrogenase (an iron sulfur flavoprotein)

ETF Dehydrogenase is an iron sulfur flavoprotein responsible for the transfer of electrons between electron transfer flavoprotein (ETF) and CoQ of the electron transport chain. We have determined the resonance Raman spectrum of this enzyme observing in the process at least seven of thirteen flavin bands in the 1100cm-1-1600 cm-1 region of the Raman spectrum. The positions of three of these bands, II, IX, and X (see Figure I and Table I for band numbering system) in ETF dehydrogenase is very similar to their positions in aqueous solution of flavins in which water is hydrogen bonded to N-1, N-5, C=0(2), C=0(4), and N-H(3) of flavin. Conversely the positions of the flavin Raman bands are considerably shifted from those of flavin in nonhydrogen bonding solvent. The positions of bands II, IX, and X are nearly identical to those in the flavoprotein glutathione reductase; x-ray structural investigations on this enzyme indicate that there is extensive hydrogen bonding between FAD and protein in this molecule. A previous study in our laboratory has demonstrated that metal complexation at N-5 and C=0(4) with either Ru or Ag produces large shifts in the positions of Raman bands II, VI, IX, and X. None of these shifts are observed in ETF dehydrogenase indicating that there is no direct inner sphere coordination of Fe to flavin. In addition to the Raman bands of flavin observed in our spectrum, we also observe one band that is in the Fe-S stretching region observed for a variety of Fe-S proteins. This band is located at 331 cm-1. The frequency of the band corresponds to the 335 cm-1 band associated with the strongest Fe-S stretching mode in the 4Fe-4S protein ferrodoxin from C. pasterianum. The observed frequency is quite different from that of the 3Fe-3S proteins such as ferrodoxin(II) from D. gigas. Finally, ETF dehydrogenase shows no loss of activity or visual evidence of photodegradation in the laser beam as most other FeS proteins do.

Resonance Raman investigation of β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase

Addition of NAD’ to FA-GPD* has been shown [l] to activate furylacyl enzyme for nucleophilic attack by phosphate; concurrently, a visible spectral red shift (X,, 344 nm to h,, 360 nm) is observed [2]. Based upon the latter observation, it has been suggested that the chemical activation results from the following conformational change from s-tram to scis in the FA chromophore. The conformational change is thought to be associated with the activation of the acyl group to nucleophilic attack by phosphate:

Identification of enzyme coupling sites with aromatic diazonium salts-a resonance raman study.

Abstract The coupling reaction of diazonium salts of aromatic compounds with the aromatic residues of proteins results in chromophoric covalent derivatives which yield strong resonance enhanced Raman spectra. The protein residues modified by these coupling reactions have been identified using the ν(NN) and ν(N-φ) vibrational bands in the resonance Raman spectra. Previous studies have established that diazoarsanilic acid couples with carboxypeptidase at tyrosine 248. The resonance Raman spectrum of arsanilazocarboxypeptidase was compared with spectra of arsanilazotyrosine and arsanilazohistidine model compounds; the results are consistent only with coupling at a tyrosine residue. This confirmation of the previously established site of modification establishes the utility of resonance Raman spectroscopy as a tool for identification of the site of covalent modification. To further investigate this approach, the diazonium salt of sulfanilamide (a site-specific reagent) was used to prepare a covalent coupling derivative of bovine carbonic anhydrase. The coupling reaction appears to have a stoichiometry of 1:1 and results in nearly complete loss of sulfanilamide binding capability and esterase activity. Comparison of the pH dependence of the resonance Raman spectra of sulfanilazocarbonic anhydrase with the spectra of sulfanilazotyrosine, sulfanilazohistidine, and sulfanilazotryptophan suggests that histidine is the site of modification of this new carbonic anhydrase derivative.

1,2-Dipalmitoyl-3-β-2-furylacryloyltriacylglycerol: a chromophoric substrate for lipoprotein lipase

Abstract We have investigated the suitability of 1,2-dipalmitoyl-3-β-2-furylacryloyltriacylglycerol as a chromophoric substrate for lipoprotein lipase from Pseudomonas fragi . Steady-state kinetic experiments of the hydrolysis of emulsions of the ester chromophore catalyzed by lipoprotein lipase indicate that the Michaelis constant ( K m ) has the same value throughout the pH range 7 to 9.5. The value of K m was determined to be 0.1 mM from appropriate reciprocal plots; this K m value is comparable to that for emulsions of other triacylglycerol substrates. Studies of the rate of hydrolysis, V max , at different pH values indicate that the reaction is faster at basic pH, suggesting base catalysis of hydrolysis. A coupled assay for glycerol formed in the hydrolysis reaction catalyzed by lipoprotein lipase suggests that the rate of hydrolysis of the furylacryloyl side chain is faster than the rate of hydrolysis of the palmitoyl side chain at position 2, indicating that the chromophoric substrate is sufficiently reactive so that the usual stereochemical preference for hydrolysis at the 3 position is preserved. When mixed liposomes of phosphatidylcholine and chromophoric substrate are incubated with lipoprotein lipase there is an initial breakdown of the liposomes with release of chromophore into solution followed by a slow hydrolysis of chromophore; this result shows that the chromophoric substrate can be useful to monitor physical changes that occur in liposomes during the hydrolysis reaction catalyzed by lipoprotein lipase.

The EH2 reduced intermediate of glutathione reductase contains oxidised flavin-while EH4 does not.

Glutathione reductase is a flavoprotein whose x-ray structure has been established. Functional data and the x-ray structure are consistent with a mechanism of reaction in which NADPH reacts with the enzyme to produce a two electron, EH2, and four electron, EH4, intermediate. The former is competent for the transfer of electrons to the substrate glutathione. Several structures are possible for the two NADPH intermediates; in order to aid in the determination of the structure of these intermediates, we have determined their resonance Raman spectra at two excitation frequencies. These studies establish that the EH2 intermediate is an oxidized flavin species while the EH4 species is not. Furthermore, the most likely structure for EH2 involves a charge transfer donation of electrons from the anion of cys-63 to the N5 position of flavin.

Kinetic methods for the study of the enzyme systems of β-oxidation☆

Abstract Kinetic methods for studying the reactions of the “general” fatty acyl CoA dehydrogenase under three sets of substrate and enzyme concentration conditions have been developed. The reaction of butyryl-CoA and electron transfer flavoprotein (ETF) can be studied either under steady-state conditions with enzyme at catalytic concentration or under single-turnover conditions with enzyme in excess. Under the latter conditions, acyl-CoA dehydrogenase acts both as a catalyst and an ultimate electron-transfer acceptor. The reductive half-reaction of butyryl-CoA and enzyme can also be studied in a separate kinetic experiment. Comparison of the pH dependences of the rate constants and isotope effects of the steady-state reaction of butyryl-CoA and ETF with the same parameters for the reductive half-reaction is consistent with a mechanism involving transfer of electrons from butyryl-CoA to ETF within a ternary complex. An alternative mechanism in which the reductive half-reaction takes place prior to the binding and reaction of ETF seems unlikely because the pH 8.5 isotope effect on the reductive half-reaction is much larger than that on the complete reaction in spite of the fact that the rates of the reactions are comparable. The pH dependence of the K m for substrate and K I for inhibitor is consistent with a mechanism for transfer of electrons within the ternary complex which involves protonation of the C group of substrates. The protonation labilizes the C-2 proton and base catalysis of the removal of the C-2 proton results in the production of the active enzyme-substrate species, namely the C-2 anion of substrate.