Protein Film Voltammetry ofRhodobacterCapsulatusXanthine Dehydrogenase

Abstract

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD(+) as the electron acceptor. R. capsulatus XDH forms an (alphabeta)(2) heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds; however, R.capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinant enzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite working electrode. Furthermore, we have determined all redox potentials of the four cofactors (Mo(VI/V), Mo(V/IV), FAD/FADH, FADH/FADH(2) and two distinct [2Fe-2S](2+/+) clusters) using a combination of potentiometric and voltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potential for the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis.

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

“…For k cat /K m , the pK a values for wild-type enzyme are 6.8 and 8.8, which are lowered only very modestly in the E232Q variant to 6.6 and 8.5. The pK a values for k cat /K m with wild-type enzyme determined here are in fair agreement with the values of 6.3 and 7.7 reported previously by Aguey-Zinsou et al (27), who monitored catalysis electrochemically (the pH dependence of k cat was not reported in this work). However, here the autocatalytic reaction with uric acid was measured directly at the electrode, which might explain the difference in the pH profile, because the latter is rather related to the ionization of uric acid (28).…”

Catalytic mechanism in successive two-step protein-film voltammetry—Theoretical study in square-wave voltammetry

“…The work [45] is seen as a most relevant example to the protein-film EEC′ mechanism. Additional biological systems complying with the elaborated EEC′ mechanism one finds by the redox transformation of Rhodobacter capsulatus Xanthine Dehydrogenase [46]. This Mo-containing enzyme undergoes two-step successive reduction of Mo VI/V , Mo V/IV by which the second redox step is catalyzed [46], thus behaving similarly to described scenario in Case B of the Results and discussion section in this manuscript.…”

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…258 This occurred for a bacterial reaction center, 287 photosystems I 288 and II, 289 bovine milk xanthine oxidase, 290 Rhodobacter capsulatus xanthine dehydrogenase 183 and DMSO reductase, [291][292][293] For adsorbed enzymes, a preliminary diagnosis of health can come from noncatalytic electrochemical studies themselves, since the conformity of the peaks to ideal predictions (section 2.1) reports on the homogeneity of reduction potentials, and the values of the measured reduction potentials can be compared to those measured under potentiometric, equilibrium titrations. Ideally, both values should agree within a few millivolts, noting that the accuracy of redox titrations is rarely better than (10 mV.…”

“…158 When the current keeps increasing linearly at high driving force instead of reaching a well defined limiting value (Figure 2F), the model in section 2.2.5.4 and ref 44 suggests that the slope at high driving force is proportional to the unknown limiting current; thus, the change in slope against substrate concentration can be used to measure the Michaelis constant but not the absolute turnover number, and this does not solve the problem of film desorption (see ref 165 for an application to Alcaligenes faecalis arsenite oxidase). When the electrode cannot be rotated, the procedure that consists in analyzing the change in peak current measured at a stationary electrode [182][183][184] leads to overestimating the value of K m (see section 2.3.2 and ref 181; this is without counting film desorption). Thus, in most cases, the values of k cat and K m cannot be determined with very high accuracy, and clearly there would be no advantage in using PFV only to measure these parameters; needless to say, we have written this review because we think that PFV proved much more useful than that.…”