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J. Biol. Chem., Vol. 278, Issue 48, 47578-47584, November 28, 2003

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Tryptophan Phosphorescence Spectroscopy Reveals That a Domain in the NAD(H)-binding Component (dI) of Transhydrogenase from Rhodospirillum rubrum Has an Extremely Rigid and Conformationally Homogeneous Protein Core^*

Jaap Broos, Edi Gabellieri, Gijs I. van Boxel¶, J. Baz Jackson¶||, and Giovanni B. Strambini

From the Department of Biochemistry and Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands, CNRS, Instituto di Biofisica, Area della Ricerca di Pisa, 56010, Pisa, Italy, and ^¶School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 °C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.

Received for publication, August 21, 2003 , and in revised form, September 11, 2003.

^* This work was supported by the Netherlands Foundation for Chemical Research, The Netherlands Organization for Scientific Research (including a travel grant to J. B.), the Italian National Research Council, the Wellcome Trust, and the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 440-121-414-5423; Fax: 440-121-414-5925. E-mail: j.b.jackson{at}bham.ac.uk.

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