Publications (7)21.11 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: The influence of para and meta substitution of benzylamine on its interaction with bovine liver mitochondrial monoamine oxidase B (MAO B) has been investigated by steady-state and reductive half-reaction anaerobic stopped-flow kinetic approaches. Steady-state kinetic properties of each benzylamine analogue suggest that para or meta substitution does not-alter the mechanistic pathway of catalysis [Husain, M., et al. (1982) Biochemistry 21, 595-600]. All analogues tested exhibited (D)k(cat) values ranging from 5.5 to 8.9 and (D)[k(cat)/K-m(amine)] values ranging from 3.3 to 8.1. (D)[k(cat)/K-m(O-2)] values of similar to 1 are observed for all substrate analogues. Values for K-d were calculated from steady-state isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Sec. 107, 1058-1060] and are in good agreement with K-s values determined from analysis of the rate of MAO B reduction as a function of benzylamine analogue concentration in reductive half-reaction experiments. A linear correlation of benzylamine analogue K-d values with the hydrophobicity parameter (pi) is observed for the para-substituted analogues where the binding affinity increases with increasing hydrophobicity of the substituent. Statistical treatment of the correlation shows a small negative contribution to binding by the van der Waals volume (V-W) of the para substituent. meta-Substituted benzylamine analogues show a decreased binding affinity with the V-W of the substituent and no correlation with the hydrophobicity value of the substituents tested. No spectral evidence was found for any flavin radical intermediates during the time course of MAO B flavin reduction in anaerobic reductive half-reduction stopped-flow experiments with any of the alpha,alpha-diprotio- or alpha,alpha-dideuteriobenzylamine analogues tested. The limiting rates of enzyme reduction exhibit large (D)k values (6.5-14.1) for all of the analogues tested. para-Substituted benzylamine analogues reduce MAO B with limiting rates that correlate with the steric influence (E(s) value) of the substituent. Statistical analysis shows the rate of MAO B reduction by para-substituted analogues to be retarded by increased values of E(s) and, with a smaller contribution, by the hydrophobicity value of the substituent. The rate of MAO B reduction by meta-substituted benzylamine analogues is essentially independent of the nature of the substituent. No evidence was found for any electronic contribution to the rate of MAO B flavin reduction by any of the analogues tested. These data demonstrate the steric orientation of the substrate to be important in the rate of amine oxidation by MAO B and that ring meta substituents favor this orientation. The relevance of these results to the proposed radical mechanism for MAO catalysis [Silverman, R. B. (1991) Biochem. Sec. Trans. 19, 201-206] is discussed.
- [Show abstract] [Hide abstract] ABSTRACT: The oxidative deamination of p-(N,N-dimethylamino)benzylamine and N-methyl-p-(N,N-dimethylamino)benzylamine by bovine liver monoamine oxidase B has been investigated by absorption spectral, steady-state, and stopped-flow kinetic studies. An absorbing intermediate with a maximum at 390 nm is observed with either analogue in turnover experiments at neutral pH and is identified as due to the formation of protonated imine as the initial product. p-(N,N-Dimethylamino)benzaldehyde is the final product formed from either substrate analogue. Anaerobic stopped-flow measurements show N-methyl-p-(N,N-dimethylamino)benzylamine to reduce enzyme-bound flavin with a limiting rate of 1.8 s-1 concurrent with the appearance of a 390-nm absorption due to protonated imine product with a limiting rate of 1.7 s-1. Both observed rates are somewhat faster than catalytic turnover (1.5 s-1). Under anaerobic conditions, the decay of protonated N-methyl-p-(N,N-dimethylamino)benzenimine is much slower than turnover (k = 4.8 x 10(4) s-1). p-(N,N-Dimethylamino)benzylamine reduces the enzyme with a limiting rate of 2.1 s-1, which is faster than catalytic turnover (1.2 s-1). Protonated imine formation is also observed with this substrate with an apparent limiting rate of 1.3 s-1. The decay of the protonated p-(N,N-dimethylamino)benzenimine absorbance is slower than catalytic turnover but faster than the rate of aldehyde formation under anaerobic conditions. Deuterium kinetic isotope effect values of approximately 10 are observed both for flavin reduction and for protonated imine formation. No isotope effect is observed for the rate of imine decay.(ABSTRACT TRUNCATED AT 250 WORDS)
- [Show abstract] [Hide abstract] ABSTRACT: Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)
- [Show abstract] [Hide abstract] ABSTRACT: A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: The influence of electrostatic forces on the formation of, and electron transfer within, transient complexes between redox proteins was examined by comparing ionic strength effects on the kinetics of the electron transfer reaction between reduced ferredoxins (Fd) and oxidized ferredoxin-NADP+ reductases (FNR) from Anabaena and from spinach, using laser flash photolysis techniques. With the Anabaena proteins, direct reduction by laser-generated flavin semiquinone of the FNR component was inhibited by complex formation at low ionic strength, whereas Fd reduction was not. The opposite results were obtained with the spinach system. These observations clearly indicate structural differences between the cyanobacterial and higher plant complexes. For the complex formed by the Anabaena proteins, the results indicate that electrostatic forces are not a major contributor to complex stability. However, the rate constant for intracomplex electron transfer had a biphasic dependence on ionic strength, suggesting that structural rearrangements within the transient complex facilitate electron transfer. In contrast to the Anabaena complex, electrostatic forces are important for the stabilization of the spinach Fd:FNR complex, and changes in ionic strength had little effect on the limiting rate constant for intracomplex electron transfer. This suggests that in this case the geometry of the initial collisional complex is optimal for reaction. These results provide a clear illustration of the differing roles that electrostatic interactions may play in controlling electron transfer between two redox proteins.
- [Show abstract] [Hide abstract] ABSTRACT: The kinetics of reduction and intracomplex electron transfer in electrostatically stabilized and covalently crosslinked complexes between ferredoxin-NADP+ reductase (FNR) and flavodoxin (Fld) from the cyanobacterium Anabaena PCC 7119 were compared using laser flash photolysis. The second-order rate constant for reduction by 5-deazariboflavin semiquinone (dRfH) of FNR within the electrostatically stabilized complex at 10 mM ionic strength (4.0 X 10(8) M-1 s-1) was identical to that for free FNR. This suggests that the FAD cofactor of FNR is not sterically hindered upon complex formation. A lower limit of approximately 7000 s-1 was estimated for the first-order rate constant for intracomplex electron transfer from FNRred to Fldox under these conditions. In contrast, for the covalently crosslinked complex, a smaller second-order rate constant (2.1 X 10(8) M-1 s-1) was obtained for the reduction of FNR by dRfH within the complex, suggesting that some steric hindrance of the FAD cofactor of FNR occurs due to crosslinking. A limiting rate constant of 1000 s-1 for the intracomplex electron transfer reaction was obtained for the covalent complex, which was unaffected by changes in ionic strength. The substantially diminished limiting rate constant, relative to that of the electrostatic complex, may reflect either a suboptimal orientation of the redox cofactors within the covalent complex or a required structural reorganization preceding electron transfer which is not allowed once the proteins have been covalently linked. Thus, although the covalent complex is biochemically competent, it is not a quantitatively precise model for the catalytically relevant intermediate along the reaction pathway.
Atlanta, Georgia, United States
- Department of Biochemistry
The University of Arizona
Tucson, Arizona, United States
- • Department of Chemistry and Biochemistry (College of Science)
- • Department of Medicine