Intramolecular electron transfer oxidases

Turowski P. N. McGuirl M. A. Dooley D. M. Intramolecular electron transfer rate between active-site copper and topa quinone in pea seedling amine oxidase. J. Biol. Chem. 1993, 268, 17680-17682. 

The chapter consists of nine sections. Sections II through VII deal with the pterin-containing molybdenum enzymes. Biochemical and model studies of molybdopterin, Mo-co, and related species are described in Section II. In Section III, we briefly survey physical and spectroscopic techniques employed in the study of the enzymes, and consider their impact upon the current understanding of the coordination about the molybdenum atom in sulfite oxidase and xanthine oxidase. Model studies are described in Sections IV and V. Section IV concentrates on structural and spectroscopic models, whereas Section V considers aspects of the reactivity of model and enzyme systems. The xanthine oxidase cycle (Section VI) and facets of intramolecular electron transfer in molybdenum enzymes (Section VII) are then treated. Section VIII describes the pterin-containing tungsten enzymes and the evolving model chemistry thereof Future directions are addressed in Section IX. 

With the exception of the recently reported DMSO reductases from bacteria (71,72), all of the enzymes of Table I contain additional redox active prosthetic groups besides Mo-co. Substrate oxidation (reduction) occurs at the molybdenum center, and electrons are removed (added) via one of the other prosthetic groups. These two processes are coupled by intramolecular electron transfer between the molybdenum center and the other redox centers of the enzyme. Results for xanthine oxidase and sulfite oxidase and approaches to modeling the coupling in sulfite oxidase are summarized below. 

Sulfite oxidase contains an oxo-molybdenum center and a 6-type cytochrome. The proposed catalytic sequence (254-256) for the enzyme is shown in Fig. 16. Oxidation of sulfite to sulfate, a two-electron process, occurs at the molybdenum center with concomitant reduction of the molybdenum from VI to IV. Electrons are removed from the enzyme by interactions of the heme of the 6-type cytochrome with exogenous cytochrome c, a one-electron process. Thus, the proposed mechanism of Fig. 16 involves two separate intramolecular electron transfers be-... 

Fig. 16. Proposed catalytic cycle of sulfite oxidase, showing the two postulated intramolecular electron transfer processes, A and B. Process B has been studied by laser flash photolysis. Adapted with permission from Kipke et al. (256). Copyright 1988, American Chemical Society.

Microcoulometric titrations of sulfite oxidase suggest that both the magnitude and the direction of the driving force for intramolecular electron transfer between the molybdenum and the iron centers of sulfite oxidase can be modulated by pH and anion concentration (78). At low pH and/or high chloride the Mo(VI) center appears to be reduced... 

An example [27] is provided by the recent determination of the rate of intramolecular electron transfer in a copper-containing amine oxidase, Cu -topaNH2/ Cu -topasQ, Eq. 26, where topa represents 3-(2,4,5-trihydroxyphenyl)-l-alanine. 

Cytochrome c oxidase (COX) is the terminal enzyme in the respiratory system of most aerobic organisms and catalyzes the four electron transfer from c-type cytochromes to dioxygen (115, 116). The A-type COX enzyme has three different redox-active metal centers A mixed-valence copper pair forming the so-called Cua center, a low-spin heme-a site, and a binuclear center formed by heme-fl3 and Cub. The Cua functions as the primary electron acceptor, from which electrons are transferred via heme-a to the heme-fl3/CuB center, where O2 is reduced to water. In the B-type COX heme-u is replaced by a heme-fo center. The intramolecular electron-transfer reactions are coupled to proton translocation across the membrane in which the enzyme resides (117-123) by a mechanism that is under active investigation (119, 124—126). The resulting electrochemical proton gradient is used by ATP synthase to generate ATP. 

Studies of intramolecular ET in oxidases provide interesting examples of how pulse radiolysis is employed to obtain insights into both (1) these enzymes respective mechanisms of action and (2) electron transfer along protein polypeptide matrices that were most probably selected by evolution (9,10, 30-32). Thus, early attempts to study the electron uptake mechanism by the blue oxidase, ceruloplasmin, showed that a diffusion-controlled decay process of the eaq in solutions of this protein is paralleled by the formation of transient optical absorptions due to electron adducts of protein residues, primarily of cystine disulfide bonds (30). The monomolecular decay of the latter absorption was found to have the same rate constant as that at which the type 1 Cu(II) absorption band was reduced. These results were interpreted as being the combined result of the high reactivity of the e q and the relatively inaccessible type 1 Cu(II) site, yielding an indirect, intramolecular electron transfer pathway from surface-exposed residues (30). 

Over the past several years, we have developed a technique that has proven extremely valuable in the study of electron transfer between redox sites in metalloproteins. We have reported kinetic studies of the reaction of cytochrome c with cytochrome c peroxidase (i-3), cytochrome oxidase (4), cytochrome bs (5, 6) plastocyanin (7), and cytochrome Ci (8). In addition, we have been able to show (9,10) that intramolecular electron transfer in cytochrome bs covalently... 

Equilibrium titration of PdNOR showed the value of Fcb was 320 mV, that of heme was - -60 mV, and those of heme c and heme ft were +310 mV and +340 mV, respectively. The rates of intramolecular electron transfer between the redox centers of NOR have been investigated using the electron backflow technique. The photolysis of CO bound to the high-spin heme results in a decrease in of the heme to its basal value, causing a transient redistribution of electrons with the other redox centers before CO rebinding occurs. Time-resolved optical spectroscopy and electrometric changes in membrane potential induced by photolysis of the CO-bound mixed-valence state of PdNOR reconstituted into proteoliposomes indicate that the pathway of electron transfer is similar to that of the heme Cu oxidases, consistent with the modeling studies discussed above. Electrons are accepted from external donors by heme c, transferred to the low-spin heme... 

The catalytic cycle is completed with re-oxidation of the molybdenum first to Mo(v), and then to Mo(vi), by intramolecular electron transfer to the cytochrome with cytochrome c serving as the external electron acceptor. Like xanthine oxidase, sulfite oxidase was among the very earliest to which XAS... 

The resting to pulsed state kinetic transition involves an increase in the rate of intramolecular electron transfer through the oxidase (i.e., from Cu to Cu,-Heme Oj) [63], Unlike the conformations involved with proton pumping, the resting and pulsed conformational states persist over minutes. Observation of the resting to pulsed state kinetic transition indicates that electfon ttansfer through the... 

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