Identification of the structural subunits required for formation of the metal centers in subunit I of cytochrome c oxidase of Rhodobacter sphaeroides.

Genetic manipulation of the aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides was used to determine the minimal structural subunit associations required for the assembly of the heme A and copper centers of subunit I. In the absence of the genes for subunits II and III, expression of the gene for subunit I in Rb. sphaeroides allowed purification of a form of free subunit I (subunit I(a)()) that contained a single heme A. No copper was present in this protein, indicating that the heme a(3)-Cu(B) active site was not assembled. In cells expressing the genes for subunits I and II, but not subunit III, two oxidase forms were synthesized that were copurified by histidine affinity chromatography and separated by anion-exchange chromatography. One form was a highly active subunit I-II oxidase containing a full complement of structurally normal metal centers. This shows that association of subunit II with subunit I is required for stable formation of the active site in subunit I. In contrast, subunit III is not required for the formation of any of the metal centers or for the production of an oxidase with wild-type activity. The second product of the cells lacking subunit III was a large amount of a free form of subunit I that appeared identical to subunit I(a)(). Since significant amounts of subunit I(a)() were also isolated from wild-type cells, it is likely that subunit I(a)() will be present in any preparation of the aa(3)-type oxidase isolated via an affinity tag on subunit I.

Suicide inactivation of cytochrome c oxidase: catalytic turnover in the absence of subunit III alters the active site.

The catalytic core of cytochrome c oxidase is composed of three subunits: I, II, and III. Subunit III is a highly hydrophobic membrane protein that contains no redox centers; its role in cytochrome oxidase function is not obvious. Here, subunit III has been removed from the three-subunit mitochondrial-like oxidase of Rhodobacter sphaeroides by detergent washing. The resulting two-subunit oxidase, subunit III (-), is highly active. Ligand-binding analyses and resonance Raman spectroscopy show that its heme a(3)-Cu(B) active site is normal. However, subunit III (-) spontaneously and irreversibly inactivates during O(2) reduction. At pH 7.5, its catalytic lifetime is only 2% that of the normal oxidase. This suicide inactivation event primarily alters the active site. Its ability to form specific O(2) reduction intermediates is lost, and CO binding experiments suggest that the access of O(2) to reduced heme a(3) is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a(3); electron transfer between the hemes is inhibited. Ligand-binding experiments and resonance Raman analysis show that increased flexibility in the structure of the active site accompanies inactivation. Cu(B) is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of Cu(B) and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the Cu(B) center via long-range interactions.