Histidine Ligand Protonation and Redox Potential in the Rieske Dioxygenases: Role of a Conserved Aspartate in Anthranilate 1,2-Dioxygenase

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• 作者：Zanna M. Beharry ; D. Matthew Eby ; Eric D. Coulter ; Rathinam Viswanathan ; Ellen L. Neidle ; Robert S. Phillips ; and Donald M. Kurtz ; Jr.
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：November 25, 2003
• 年：2003
• 卷：42
• 期：46
• 页码：13625 - 13636
• 全文大小：377K
• 年卷期：v.42,no.46(November 25, 2003)
• ISSN：1520-4995

文摘

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation ofanthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion tothe diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which acceptselectrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), aconserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led tocomplete inactivation, which was proposed to arise from elimination of a productive intersite electrontransfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837].Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate alsoresulted in enzymes that were completely inactive over a wide pH range despite retention of the hexamericquaternary structure and iron center occupancy. The Rieske center reduction potential of this variant wasmeasured to be ~100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-typeAntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrumwhich resembled that of the D218 variants at neutral pH. These results support a role for this aspartate inmaintaining the protonated state and reduction potential of the Rieske center. Both the wild-type andD218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electrontransfer pathway or in productive substrate gating of the Rieske center reduction potential. However,since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by theD218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near themononuclear iron site.