Volume 33.5, number 1, 13-17 FEBS 13280 0 1993 Federation of European Biochemical Societies 00145793/93/%6.00 Cytochrome d axial ligand of the M-type terminal quinol oxidase from November 1993 Escherichia coli Motonari TsubakP*, Tadayuki Unob, Hiroshi Hori”, Tatsushi Mogid, Yoshifumi Nishimura”, Yasuhiro Anrakud zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO aDepartment of Ltfe Science, Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-12, Japan bFaculty of Pharmaceutical Sciences, University of Tokushima, Shomachi, Tokushima, Tokushima 770, Japan ‘Department of Biophysical Engineering, Faculty of Engineering Science, Toyonaka, Osaka 560, Japan ‘Department of Plant Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan ‘Graduate School of Integrated Science, Yokohama City University, Seto, Kanazawa-ku, Yokohama, Kanagawa 236, Japan Received 17 September 1993 Using various spectroscopic techniques, we studied the structure of the dioxygen reduction site of the bd-type terminal quinol oxidase in the aerobic respiratory chain of Escherichia colt’. Resonance Raman and FT-IR spectroscopies identified the v(Fe*+-CO) and v(C-0) stretching frequencies at 471 and 1980.7 cm-‘, respectively, at the cytochrome d center of the dithionite-reduced CO-bound enzyme. The CO ligation in the cytochrome bd complex is considerably different from those of the heme-copper terminal oxidases. Anaerobic addition of NO to the air-oxidized enzyme caused an exchange of cytochrome d-bound dioxygen with NO leading to an appearance of cytochrome d-NO EPR signal. But there is no superhyperhne structure originating from the cytochrome d proximal 14N ligand in the central resonance of the NO EPR signal. These results suggest that cytochrome d axial ligand of the cytochrome bd complex is likely a histidine residue in an anomalous condition or other than a histidine residue and, therefore, the molecular structure around the dioxygen-binding site is different from that of the heme-copper terminal oxidases. Cytochrome bd complex; Cytochrome d axial ligand; Resonance Raman; Fe-CO bond; EPR; Nitric oxide 1. INTRODUCTION The cytochrome bd complex is one of terminal quinol oxidases in the aerobic respiratory chain of Escherichiu co/i and expressed predominantly under low oxygen pressure. The cytochrome bd complex has a higher af- finity for molecular oxygen and more resistant to respi- ratory inhibitors such as cyanide and azide than the cytochrome bo complex, an alternative quinol oxidase comprising the heme-copper binuclear center for the dioxygen reduction [l]. This enzyme is encoded by the cydAB genes [2] and consist of two polypeptides; sub- unit I (58 kDa) and subunit II (43 kDa) [3,4]. Based on optical spectroscopic properties, it is claimed, there are three types of cytochrome species associated with this complex; cytochrome b5s8, cytochrome bsg5, and cyto- chrome d [5,6]. Subunit I contains cytochrome bsss that shows the a and p peaks at 562 and 532 nm, respec- tively, in the reduced state at room temperature [7] and is most likely the ubiquinol-8 oxidation site [8]. Cyto- chrome b,,, is an unusual b-type cytochrome exhibiting its a andp bands at 595 and 562 nm, respectively, in the reduced minus oxidized difference spectrum [6]. Cyto- chrome d has a chlorin chromophore (heme D) [9], ex- hibiting a characteristic absorption maximum at 628 nm *Corresponding author. Fax: (81) 7915-8-0189. in the fully reduced state and is a primary exogenous ligand binding site [3,10]. In the air-oxidized condition, cytochrome d is actually in the reduced state and coor- dinates a molecular oxygen zyxwvutsrqponmlkjihgfedcbaZYX [l zyxwvutsrqponmlkjihgfedc 11. On the other hand, EPR studies on the cytochrome bd complex revealed that there are a total of four heme species in the air- oxidized state besides cytochrome d which exists as a Fe2+-O2 diamagnetic EPR-invisible state: two high-spin heme components (one axial and one rhombic species) and two minor low-spin heme components at g, ~3.3 and g, = 2.5. Although the assignment for these EPR signal is controversial, there seems a consensus that the g, = 2.5 species represents a subpopulation of cyto- chrome d [10,12-141. Here we report results of a combined study using resonance Raman, FT-IR and EPR spectroscopies for the purified cytochrome bd complex from E. cob. We present a line of evidence to suggest that the proximal ligand of cytochrome d component of the cytochrome bd complex is likely different from a usual histidine ligand. 2. MATERIALS AND METHODS 2.1. E. coli strain and growth conditions E. coli strain ST4533 (W3092 dcyo-Km’ cyd+ recA srlA::TnlO) which lacks the entire cytochrome bo operon was used in this study. Cells were grown at 37°C in a rich medium [15] supplemented with Published by Elsevier Science Publishers B. K 13