Abstract
Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction by dithionite, and an N-quinol form that was generated by reduction by substrate. It was previously shown that on reduction by substrate, an amino group displaces a carbonyl oxygen on TTQ, and that this significantly alters the rate of its oxidation by azurin (Hyun, Y-L., and Davidson V. L. (1995) Biochemistry 34, 12249-12254). To determine the basis for this change in reactivity, comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper of azurin were performed. The reaction of the O-quinol exhibited values of electronic coupling (H(AB)) of 0.13 cm(-1) and reorganizational energy (lambda) of 1.6 eV, and predicted an ET distance of approximately 15 A. These results are consistent with the ET event being the rate-determining step for the redox reaction. Analysis of the reaction of the N-quinol by Marcus theory yielded an H(AB) which exceeded the nonadiabatic limit and predicted a negative ET distance. These results are diagnostic of a gated ET reaction. Solvent deuterium kinetic isotope effects of 1.5 and 3.2 were obtained, respectively, for the ET reactions from O-quinol and N-quinol AADH indicating that transfer of an exchangeable proton was involved in the rate-limiting reaction step which gates ET from the N-quinol, but not the O-quinol. These results are compared with those for the ET reactions from another TTQ enzyme, methylamine dehydrogenase, to amicyanin. The mechanism by which the ET reaction of the N-quinol is gated is also related to mechanisms of other gated interprotein ET reactions.
Highlights
Transient kinetic studies yielded significantly different values for the limiting first-order rate constant for the oxidation of reduced aromatic amine dehydrogenase (AADH) by azurin depending upon whether AADH had been reduced chemically with dithionite, or with the substrate tyramine [9]
Different reaction rate constants were obtained depending upon whether methylamine dehydrogenase (MADH) was reduced chemically with dithionite, or with the substrate methylamine [11, 12]. Thermodynamic analysis of these redox reactions indicated that the oxidation of dithionite-reduced quinol (O-quinol) TTQ by amicyanin was rate-limited by the electron transfer (ET) event [13, 14], but that the oxidation of the substrate-reduced aminoquinol (N-quinol) TTQ by amicyanin was a gated ET reaction [11, 12]
We present comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper in azurin (Fig. 1)
Summary
Purifications of AADH [1] and azurin [8] from A. faecalis (IFO 14479) were as described previously, and protein concentrations were calculated from previously determined extinction coefficients [1, 15]. The experimental procedures for the rapid mixing experiments were as described previously [9] for the reactions of the O-quinol AADH with azurin. For the reactions of the N-quinol, it was necessary to use anaerobic conditions This is because the N-quinol AADH exhibited significant reactivity with O2, and during the incubation times before mixing some conversion of N-quinol to N-semiquinone occurred. With solutions which contained protein samples, H2O was completely exchanged for D2O by repeated ultrafiltration using Centriprep (Amicon Inc., Beverly, MA) concentrators. After solvent exchange, these protein solutions were incubated overnight in the buffered D2O at 10 °C to ensure the complete exchange of all solvent exposed titratable hydrogen ions for deuterium ions. The oxidation-reduction midpoint potential (Em) value of azurin was determined by spectrochemical titration as described previously for the determination of the Em value of amicyanin [22]
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