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Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase.

ABSTRACT: Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2- to Cl- and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (approximately 4.7 x 10(3) micromol of ClO2- min(-1) mg(-1) subunit) and nearly stoichiometric in heme; further, it shares spectroscopic and physicochemical features with chlorite dismutases previously isolated from three organisms. A careful study of the enzyme's steady state kinetics has been carried out. ClO2- consumption and O2 release rates were measured, yielding comparable values of kcat (4.5 x 10(5) min(-1)), K(m) (approximately 215 microM), and kcat/Km (3.5 x 10(7) M(-1) s(-1) via either method (4 degrees C, pH 6.8; all values referenced per heme-containing subunit). ClO2-:O2 stoichiometry exhibited a 1:1 relationship under all conditions measured. Though the value of kcat/Km indicates near diffusion control of the reaction, viscosogens had no effect on k(cat)/K(m) or V(max). The product O2 did not inhibit the reaction at saturating [O2], but Cl- is a mixed inhibitor with relatively high values of KI (225 mM for enzyme and 95.6 mM for the enzyme-substrate complex), indicating a relatively low affinity of the heme iron for halogen ions. Chlorite irreversibly inactivates the enzyme after approximately 1.7 x 10(4) turnovers (per heme) and with a half-life of 0.39 min, resulting in bleaching of the heme chromophore. The inactivation K(I) (K(inact)) of 166 microM is similar in magnitude to Km, consistent with a common Michaelis complex on the pathway to both reaction and inactivation. The one-electron peroxidase substrate guaiacol offers incomplete protection of the enzyme from inactivation. Mechanisms in keeping with the available data and the properties of other well-described heme enzymes are proposed.

SUBMITTER: Streit BR

PROVIDER: S-EPMC3742725 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase.

Streit Bennett R BR DuBois Jennifer L JL

Biochemistry 20080419 19

Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2- to Cl- and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (approximately 4.7 x 10(3) micromol of ClO2- min(-1) mg(-1) subunit) and nearly stoichiometric in heme; furthe ...[more]

PMID: 18422344

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Project description:Chlorite dismutase catalyzes O(2) release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady-state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3-11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid-base transition was observed by UV/visible and resonance Raman (rR) spectroscopy with a pK(a) of 8.7, 2-3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five-coordinate high-spin Fe(III) to a mixed high/low-spin ferric hydroxide, as confirmed by rR spectroscopy. The two Fe-OH stretching frequencies are quite low, consistent with a weak Fe-OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe-OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond-donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pK(a) approximately 6.5) form also appears to act as the active-site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady-state profile for the rate of chlorite decomposition is characterized by these same pK(a) values. The five-coordinate high-spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum k(cat) = 2.0(+/-0.6) x 10(5) s(-1), k(cat)/K(M) = 3.2(+/-0.4) x 10(7) M(-1) s(-1), pH 5.2, 4 degrees C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions.

Dataset Information

Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase.

Publications

Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase.

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