Fig. 1: Physiological and biochemical links between CydDC activity and cytochrome bd biogenesis.
From: Dissecting the conformational complexity and mechanism of a bacterial heme transporter
a, E. coli growth-complementation studies. Plasmid encoded cytochrome bd variants were pcydABX, cyt. bd-I; pappCBX, cyt. bd-II. Plasmid encoded CydDC variants were pcydDC, wild-type; pE500Q, E->Q exchange at position 500 of CydC; pH85A, H->A exchange at position 85 of CydC. Control plasmid was pcontrol, empty pET17 vector. Data are presented as mean values ±s.d. (n = 3 biological replicates). b, Reduced-minus-oxidized UV-vis spectra of membrane fractions from strains with restored and/or impaired growth phenotypes. c, Oxygen reductase activity of membranes from strains with growth-active phenotypes. Aurachin D (Aur. D) induced inhibition of oxygen consumption indicates for cytochrome bd specific oxygen reductase activity. Data are presented as mean values ±s.d. (n = 3 biological replicates). Significance was assessed based on a paired two-tailed Student’s t-test. Rel., relative. d, Stimulation of ATP hydrolysis activity (malachite green phosphate assay) of CydDC. Significance was assessed based on a paired two-tailed Student’s t-test. All presented ATP hydrolysis data are corrected for background activity in the absence of substrate candidates. Data are presented as mean values ± s.d. (n = 3 assay technical replicates). CSSC, cystine; GSSG, glutathione disulfide; P-IX, protoporphyrin IX. e, Titration experiments for the determination of Michaelis constants (KMapp) for ATP (91.9 ± 9.7 µM) and heme (79.7 ± 1.2 nM). All presented ATP hydrolysis data are corrected for background activity in the absence of substrate candidates. Data are presented as mean values ± s.d. (n = 3 assay technical replicates). f, Working model of the physiological role of CydDC based on independent physiological and biochemical data generated in this study.
