Fourier transform infrared (FTIR) and step-scan time-resolved FTIR spectroscopies reveal a unique active site in cytochrome caa3 oxidase from Thermus thermophilus.
Fourier transform infrared (FTIR) and step-scan time-resolved FTIR difference spectra are reported for the [carbonmonoxy]cytochrome caa(3) from Thermus thermophilus. A major C-O mode of heme a(3) at 1958 cm(-1) and two minor modes at 1967 and 1975 cm(-1) (7:1:1) have been identified at room temperature and remained unchanged in H(2)O/D(2)O exchange. The observed C-O frequencies are 10 cm(-1) higher than those obtained previously at 21 K (Einarsdóttir, O., Killough, P. M., Fee, J. A., and Woodruff, W. H. (1989) J. Biol. Chem. 264, 2405-2408). The time-resolved FTIR data indicate that the transient Cu(B)(1+)-CO complex is formed at room temperature as revealed by the CO stretching mode at 2062 cm(-1). Therefore, the caa(3) enzyme is the only documented member of the heme-copper superfamily whose binuclear center consists of an a(3)-type heme of a beta-form and a Cu(B) atom of an alpha-form. These results illustrate that the properties of the binuclear center in other oxidases resulting in the alpha-form are not required for enzymatic activity. Dissociation of the transient Cu(B)(1+)-CO complex is biphasic. The rate of decay is 2.3 x 10(4) s(-1) (fast phase, 35%) and 36.3 s(-1) (slow phase, 65%). The observed rate of rebinding to heme a(3) is 34.1 s(-1). The implications of these results with respect to the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases are discussed.
Pinakoulaki
Eftychia
E
Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece and the Paul Scherrer Institut, Life Sciences, OSRA/008, CH-5232 Villigen PSI, Switzerland.
Soulimane
Tewfik
T
Varotsis
Constantinos
C
D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D003574:Cytochrome c Group / Q000737:chemistry*; D045264:Cytochromes a; D045266:Cytochromes a3; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D008027:Light; D011485:Protein Binding; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013696:Temperature; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology* / Q000378:metabolism
D003574:Cytochrome c Group; D045266:Cytochromes a3; C435384:cytochrome caa(3); D006418:Heme; D045264:Cytochromes a; D003576:Electron Transport Complex IV