a, A schematic of the respiratory chain of F. johnsoniae. b, UV–visible spectrum and SDS–PAGE of the membranes from F. johnsoniae. Left, the difference spectrum of the membranes of F. johnsoniae, obtained from the spectrum of the air-oxidized membranes and the spectrum after reduction with dithionite. The wavelengths associated with the haem peaks are 605 nm, 560 nm, 552 nm and a broad peak at 630 nm for haems a, b, c and d, respectively. Right, the SDS–PAGE with the membranes followed by staining the gel for haems shows bands corresponding to the cytochrome subunits ActA (48 kDa) and ActE (20 kDa) of ACIII but no bands corresponding to the cytochrome subunit (around 35 kDa) from the cbb 3 oxidase. c, The gene arrangement for the ACIII and the cytochrome oxidase aa 3 genes in the F. johnsoniae genome. The genes for the subunits I and II from cyt aa 3 oxidase are found immediately downstream of those for the act genes of the ACIII. Two different versions of subunit III are denoted as vI and vII. d, UV–visible spectra of the reduced and oxidized forms of the supercomplex in detergent and SMA nanodiscs. The dithionite reduced form of the samples is represented in red and shows the peaks for haem c at 524 nm and 552 nm and those for haem a at 443 nm and 605 nm. e, Pyridine haemochrome assay of the ACIII–cyt aa 3 supercomplex in SMA nanodiscs. Plotted is the reduced-minus-oxidized difference spectrum of the pyridine haemochromes of the sample. Peaks at 520 nm and 550 nm are associated with haem c and the peak at 590 nm is associated with haem a. Quantification from the spectrum shows a ratio of 10.6:1 between haem c and haem a, which translates into a 3:2 ratio between ACIII and cyt aa 3 assuming 7 haem c per ACIII and 2 haem a per cyt aa 3 . Data in b are representative of two independent experiments with similar results, and data in d and e are representative of six independent experiments with similar results.