Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I.

Abstract

The two-subunit cytochrome c oxidase fromParacoccus denitrificans has been sequentially digested with chymotrypsin and Staphylococcus aureus V8 protease. The smaller subunit of the enzyme (apparent Mr 32,000) was split into numerous peptides that were removed by anion-exchange HPLC. The larger subunit was only digested to a limited extent (from an apparent Mr 45,000 to M, 43,000), and the spectral properties were preserved relative to the native enzyme (a reduced minus oxidized difference spectrum with maxima at 447 and 607 nm in the Soret and a region, respectively). As judged from CO-reduced spectra this proteolytically digested, one-fragment oxidase was found to contain an equal amount of cytochromes a and a3. The enzymatic activity with reduced cytochrome c as substrate in the presence of Triton X-100 proceeded with equal affimity (apparent K. = 0.5-1.0 pM) and with a V__ of =20% (40 s-1) of that found with the native

Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent

“…Results of these studies indicate that the high-affinity binding site is located in subunit II, with some of the nuclear subunits constituting part of the interaction domain (Bisson & Montecucco, 1982;Kadenbach & Stroh, 1984). In contrast, the results of Muller et al (1988) suggest that the Km for cytochrome c is unaltered after proteolytic digestion of subunit II in Paracoccus denitrificans cytochrome oxidase. The location and significance of the low-affinity site have yet to be determined.…”

Cytochrome c oxidase: understanding nature's design of a proton pump

“…Spectroscopically, cytochrome a and CuA interact only weakly and are often treated as independent electron acceptors. On the basis of spectroscopic evidence as well as sequence homology data, it is now generally agreed that these redox-active metal centers are located in subunits I and II (Mueller et al, 1988). Specifically, it has been suggested that cytochrome a, cytochrome a3, and CuB reside in subunit I while CuA is associated with subunit II [for a model, see Holm et al (1987)].…”

X-ray Absorption Studies on the Mixed-Valence and Fully Reduced Forms of the Soluble CuA Domains of Cytochrome c Oxidase

“…The heme−copper oxidases are members of a superfamily of proton-pumping metalloenzymes found in both prokaryotic and eukaryotic organisms, which couple the energy released from the 4e - reduction of dioxygen to water to the formation of a proton motive force across the membrane which can be used for the synthesis of ATP. , The most-widely studied is the class of oxidases found in mammalian mitochondria, yeasts, and a number of bacterial systems such as Thermus thermophilus, Rhodobacter spheroides, , and Paracoccus denitrificans. − This branch of the superfamily utilizes cytochrome c as the electron-donating substrate and contains three redox centers: Cu A (thiolate-bridged binuclear mixed-valence center), heme a (6-coordinate low spin), and the dioxygen-binding heme a 3 −Cu B (magnetically coupled, S = 2 binuclear center). A second major branch of the family, exemplified by the cytochrome bo 3 and aa 3 -600 oxidases of Escherichia coli 1,2,9 and Bacillus subtilis , , respectively, uses ubiquinol as its reducing substrate, and in these cases, the Cu A -binding site is absent.…”