LETTERS Detection of a Photostable Five-Coordinate Heme a 3 -Fe-CO Species and Functional Implications of His384/r10 in CO-Bound ba 3 -Cytochrome c Oxidase from Thermus thermophilus Takehiro Ohta, ‡ Eftychia Pinakoulaki, † Tewfik Soulimane, § Teizo Kitagawa, ‡ and Constantinos Varotsis* ,† Department of Chemistry, UniVersity of Crete, 71409 Heraklion, Crete, Greece, Center for IntegratiVe Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki, Aichi 444-8585 Japan, and Paul Scherrer Institute, Life Sciences, OSRA/008,CH-5232 Villigen PSI, Switzerland ReceiVed: February 18, 2004; In Final Form: March 22, 2004 Resonance Raman (RR) spectra are reported for the fully reduced carbon monoxy derivative of ba 3 -cytochrome c oxidase from Thermus thermophilus. The RR spectra show the formation of a photolabile six-coordinate heme-CO and a photostable five-coordinate heme Fe-CO species. The latter species is formed by the cleavage of the proximal heme Fe-His384 bond and is the first five-coordinate Fe-CO species detected in heme- copper oxidases. The frequency of the Fe-CO species observed at 526 cm -1 correlates with either the C-O stretching modes observed at 1967 or 1982 cm -1 and lie on the correlation line of ν(Fe-CO) vs ν(C-O) for all known five-coordinate heme Fe-CO complexes. The loss of intensity of the heme Fe-His384 mode observed at 193 cm -1 in the photostationary CO-bound spectra is attributed to the loss of the non-hydrogen bonded heme Fe-His384‚‚‚Gly359 conformer. Taken together, the data indicate that the environment of the ruptured His384 that is a part of the Q-proton pathway and leads to the highly conserved among all heme- copper oxidases, H 2 O pool, is disrupted upon CO binding to heme a 3 . Introduction The chemistry of carbon monoxide (CO) and nitric oxide (NO) with the active heme centers of biological sensors that carry out important roles in biological signaling in eukaryotic and prokaryotic organisms, and with the binuclear center of heme-copper oxidases is of profound relevance. 1-5 CO has been shown to stimulate guanylate cyclase (sGC) activity and is generally believed to activate the protein in a manner similar to NO. 1-3 Activation of sGC brought about by NO is due to NO coordination to the heme followed by rupturing of the heme Fe-His bond, yielding a five-coordinate (5C) heme-NO species. 2,3 On the other hand, CO binding to sGC forms both six-coordinate (6C) and 5C heme-CO complexes. 1 Although the inhibition of heme-copper oxidases by NO plays a physiological role in controlling mitochondrial O 2 consumption, the inhibition of the respiratory enzymes by CO and the molecular mechanisms of the origin of ligand specificity are open questions. 4 The ba 3 -cytochrome c oxidase from the gram-negative thermophilic eubacterium Thermus thermophilus couples the reduction of dioxygen to proton translocation across the inner bacterial membrane and, in contrast to the eucaryotic heme- copper oxidases, catalyzes the reduction of NO to N 2 O. 6,7 The enzyme contains a homodinuclear copper complex (Cu A ), one * To whom correspondence should be addressed: E-mail: varotsis@ edu.uoc.gr. † University of Crete. ‡ Okazaki National Research Institutes. § Paul Scherrer Institute, Life Sciences. © Copyright 2004 by the American Chemical Society VOLUME 108, NUMBER 18, MAY 6, 2004 10.1021/jp049259k CCC: $27.50 © 2004 American Chemical Society Published on Web 04/10/2004