Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase W. Anthony Oertling a, * , Charisa D. Cornellison a,1 , Nathan R. Treff a,2 , Junji Watanabe a,3 , Michelle A. Pressler b,4 , Jeanne R. Small a,5 a Department of Chemistry and Biochemistry, 226 Science Building, Eastern Washington University, Cheney, WA 99004-2440, USA b Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA Received 4 November 2006; received in revised form 8 December 2006; accepted 12 December 2006 Available online 21 December 2006 Abstract We report a protein conformational change following carbon monoxide photodetachment from fully reduced bovine cytochrome c oxidase that is hypothesized to be associated with changes in ligand mobility through a dioxygen access channel in the protein. Although not resolved by earlier photoacoustic or optical studies on this adduct, utilization of slightly lower temperatures revealed a process with a kinetic lifetime of about 70 ns at 10 °C. We measure an enthalpy change of about 8 kcal/mol in 0.050 M HEPES buffer that becomes less endothermic (DH  2 kcal/mol) at higher ionic strength. The volume contraction of about 0.7 mL/mol associated with the process almost doubles in higher ionic strength buffer systems. Measurements of samples in phosphate buffer systems are similar and appear to display the same subtle ionic strength dependence. Both the isolation of this photoacoustic signal component and the possible depen- dence on ionic strength of the thermodynamic parameters derived from its analysis appear analogous to and consistent with prior photoacoustic results monitoring CO photodetachment from the camphor complex of cytochrome P-450. Accordingly, we consider a similar model in which a conformational change results in movement of an exposed charged group or groups towards the interior of the protein, out of contact with solvent, as in the closing of a salt bridge. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Photoacoustics; CO photodetachment; Fully reduced; Cytochrome c oxidase; Bovine cytochrome aa 3 1. Introduction In mitochondria and numerous bacteria, the energy needed to drive respiratory phosphorylation of adenosine diphosphate is stored as a proton gradient. Three metallo- protein complexes pump protons out of the matrix across the inner membrane of mitochondria or across the plasma membrane of bacteria. Energy for this active transport is provided by the redox reactions of the electron transport chain. As the last of these proton pumps, and the terminal component of the respiratory chain, cytochrome oxidase binds molecular oxygen and catalyzes its reduction to water by using electrons from foodstuff delivered by mobile carriers ubiquinol or cytochrome c. In the case of cyto- chrome c oxidases (CcO), electrons are transferred through the protein via transition metal cofactors labeled Cu A , a, 0162-0134/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.12.003 * Corresponding author. Tel.: +1 509 359 7476; fax: +1 509 359 6973. E-mail address: WOertling@ewu.edu (W. Anthony Oertling). 1 Present address: Corixa Corporation, 1900 9th Avenue, Suite 1100, Seattle, WA 98101, USA. 2 Present address: University of Wisconsin, Madison School of Medi- cine, Department of Surgery, CSC H5/301, 600 Highland Avenue, Madison, WI 53792, USA. 3 Present address: University of California, Department of Medicine/ Division of Cardiology, Atherosclerosis Research Units, MRL Room 3535, 675 Charles & Young Dr., Los Angeles, CA 90095, USA. 4 Present address: Thermo-Electron Corp., 1201 E. Wiley Road, Suite 160, Schaumburg, IL 60173, USA. 5 Present address: Department of Chemistry – CMDITR, University of Washington, Box 351700, Seattle, WA 98195-1700, USA. www.elsevier.com/locate/jinorgbio Journal of Inorganic Biochemistry 101 (2007) 635–643 JOURNAL OF Inorganic Biochemistry