Menaquinone as Well as Ubiquinone as a Bound Quinone Crucial for Catalytic Activity and Intramolecular Electron Transfer in Escherichia coli Membrane-bound Glucose Dehydrogenase

Golam Mustafa,Catharina T Migita,Yoshinori Ishikawa,Kazuo Kobayashi,Seiichi Tagawa,Mamoru Yamada

doi:10.1074/jbc.m804938200

Golam Mustafa, Catharina T Migita + Show 4 more

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https://doi.org/10.1074/jbc.m804938200

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Abstract

Escherichia coli membrane-bound glucose dehydrogenase (mGDH), which is one of quinoproteins containing pyrroloquinoline quinone (PQQ) as a coenzyme, is a good model for elucidating the function of bound quinone inside primary dehydrogenases in respiratory chains. Enzymatic analysis of purified mGDH from cells defective in synthesis of ubiquinone (UQ) and/or menaquinone (MQ) revealed that Q-free mGDH has very low levels of activity of glucose dehydrogenase and UQ2 reductase compared with those of UQ-bearing mGDH, and both activities were significantly increased by reconstitution with UQ1. On the other hand, MQ-bearing mGDH retains both catalytic abilities at the same levels as those of UQ-bearing mGDH. A radiolytically generated hydrated electron reacted with the bound MQ to form a semiquinone anion radical with an absorption maximum at 400 nm. Subsequently, decay of the absorbance at 400 nm was accompanied by an increase in the absorbance at 380 nm with a first order rate constant of 5.7 x 10(3) s(-1). This indicated that an intramolecular electron transfer from the bound MQ to the PQQ occurred. EPR analysis revealed that characteristics of the semiquinone radical of bound MQ are similar to those of the semiquinone radical of bound UQ and indicated an electron flow from PQQ to MQ as in the case of UQ. Taken together, the results suggest that MQ is incorporated into the same pocket as that for UQ to perform a function almost equivalent to that of UQ and that bound quinone is involved at least partially in the catalytic reaction and primarily in the intramolecular electron transfer of mGDH.

Highlights

Topological analysis of membrane-bound glucose dehydrogenase (mGDH) revealed a unique structure consisting of an N-terminal hydrophobic domain with five membrane-spanning segments and a large C-terminal domain residing in the periplasm [15], which contains pyrroloquinoline quinone (PQQ) as a coenzyme and Ca2ϩ- or Mg2ϩ-binding sites in a superbarrel structure, conserved in quinoproteins [15,16,17,18,19]
It has been demonstrated that mGDH has two UQ-binding sites, one (QI) for bound UQ and the other (QII) for bulk UQ [26], which is near the membrane surface rather than in the hydrophobic interior [27], and that intramolecular electron transfer following the catalytic reaction occurs from PQQH2 directly to UQ in the QII site or via bound UQ
Our recent studies revealed that eaϪq produced by pulse radiolysis caused a rapid reduction of bound UQ followed by intramolecular electron transfer to PQQ in mGDH and suggested that the two redox centers are closely located at a distance of 11–13 Å [28] and that Asp-466 and Lys-493 are involved in proton donation to the semiquinone anion radical of bound UQ and in electron transfer from bound UQ to PQQ, respectively [25]

Summary

Introduction

We provided the first evidence that the primary dehydrogenase in respiratory chains utilizes both MQ and UQ as a bound Q and suggest that bound MQ occurs in a fashion similar to that of bound UQ in the mGDH molecule and functions as an electron acceptor from PQQ. Enzymatic Comparison of Q-free and MQ-bearing mGDHs with UQ-bearing mGDH—Enzymatic activity of purified mGDH is generally evaluated either with PMS or UQn with a short isoprenoid chain as an electron acceptor following glucose oxidation [15, 24].

Results

Conclusion