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Biophysical Journal 86:3230-3240 (2004)
© 2004 The Biophysical Society

Noninvasive Auto-Photoreduction Used as a Tool for Studying Structural Changes in Heme-Copper Oxidases by FTIR Spectroscopy

Karin Bettinger, Alexander Prutsch, Karsten Vogtt and Mathias Lübben

Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germany

Correspondence: Address reprint requests to Mathias Lübben, Tel.: +49-234-32-24465; Fax: +49-234-32-14626; E-mail: luebben{at}bph.rub.de.

We demonstrate an efficient Fourier transform infrared (FTIR) spectroscopic method, termed "auto-photoreduction," that uses anaerobic photo-induced internal electron transfer to monitor reaction-initiated changes of heme-copper oxidases. It can be applied without the use of either expensive electrochemical equipment, or caged compounds, which cause significant background signals. At high irradiation power, carbon monoxide is released from high-spin heme a of cytochrome c oxidase and heme o from cytochrome bo3. Photochemistry is initiated at wavelengths <355 nm, and the photochemical action spectrum has a maximum of 290 nm for cytochrome bo3, which is consistent with the possible intermediate involvement of tyrosinate or an activated state of tyrosine. We propose that the final electron donors are proton channel water molecules. In the pH range of 4–9, the noninvasive auto-photoreduction method yields highly reproducible FTIR redox difference spectra within a broad range, resolving a number of vibrational changes outside the amide I region (1600–1640 cm–1). Furthermore, it provides details of redox-induced changes in the spectral region between 1600 and 1100 cm–1. The auto-photoreduction method should be universally applicable to heme proteins.






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Copyright © 2004 by the Biophysical Society.