Alternative Initial Proton Acceptors for the D Pathway of Rhodobacter sphaeroides Cytochrome c Oxidase

Abstract

In order to characterize protein structures that control proton uptake, forms of cytochrome c oxidase (CcO) containing a carboxyl or a thiol group in line with the initial, internal waters of the D pathway for proton transfer have been assayed in the presence and absence of subunit III. Subunit III provides approximately half of the protein surrounding the entry region of the D pathway. The mutant N139D-D132N contains a carboxyl group 6Å within the D pathway and lacks the normal, surface-exposed proton acceptor, Asp-132. With subunit III, the steady-state activity of this mutant is slow but once subunit III is removed its activity is the same as wild-type CcO lacking subunit III (∼1800 H + s -1 ). Thus, a carboxyl group ∼25% within the pathway enhances proton uptake even though the carboxyl has no direct contact with bulk solvent. Protons from solvent apparently move to internal Asp-139 through a short file of waters, normally blocked by subunit III. Cysteine-139 also supports rapid steady-state proton uptake, demonstrating that an anion other than a carboxyl can attract and transfer protons into the D pathway. When both Asp-132 and Asp/Cys-139 are present, the removal of subunit III increases CcO activity to rates greater than that of normal CcO due to simultaneous proton uptake by two initial acceptors. The results show how the environment of the initial proton acceptor for the D pathway in these CcO forms dictates the pH range of CcO activity, with implications for the function of Asp-132, the normal proton acceptor.The aa 3 -type cytochrome c oxidase complex (CcO) is the final member of the electron transfer chain of oxidative phosphorylation in the inner membrane of mitochondria and in the cytoplasmic membrane of many bacteria. In order to generate a voltage gradient across these membranes, CcO transfers electrons from its outer (positive) surface and protons from its inner (negative) surface to a buried heme-Cu active site where the charges combine as O 2 is reduced to water (1-3). As a proton pump, CcO also uses the energy of O 2 reduction to transfer protons completely through the protein, across the membrane (3-6). The foursubunit aa 3 -type CcO of the bacterium Rhodobacter sphaeroides is one of the popular, mutable models of the catalytic core of mitochondrial CcO (7,8).Cytochrome c oxidase (CcO) takes up the protons necessary for catalysis via two long pathways, the D and K paths, leading from the inner surface of the complex to the buried active site (Fig. 1) (2,4,9,10). Protons are introduced into the D pathway by Asp-132 of subunit I (R. sphaeroides numbering) and transferred 26Å to Glu-286, near the active site, through a series of hydrogen-bonded water molecules (11)(12)(13)(14). From Glu-286, protons branch to O 2 reduction intermediates in the active site or to the site of proton pumping (15-17). The K pathway begins far from the entry region of the D pathway, at Glu-101 of subunit II (18). The K pathway transfers protons primarily via protein side chains to the cross-linked Tyr-288/His...

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

“…As described above, results from earlier studies have shown that introduction of negative charges near the D pathway orifice in the D132N/A mutant oxidases could restore proton pumping and accelerate proton uptake (18,19,39). These data were interpreted to suggest that slowed proton uptake in the D132N single mutant is caused by the removal of negative charge (40).…”

“…Proton uptake in the D132A CytcO also could be partly restored upon removal of SUIII, which eliminates a significant part of the residues surrounding the D-pathway orifice and presumably allows water molecules to access the proton pathway (38). Even more dramatic effects were observed for the N139C/D132N and N139D/D132N double-mutant CytcOs, where removal of SUIII resulted in restoration of full CytcO activity (19). The data from the present study indicate that the proton-uptake rate through the D pathway could be accelerated by a factor of 5,000, to reach the maximum observed value (∼100 μs), upon introduction of a single structural mutation, N139T in the D132N mutant CytcO.…”

“…Rapid proton uptake and pumping could be partly restored by introduction of an Asp at the site of Asn-139, located ∼7 Å from Asp-132 (N139D/ D132N double mutation) (18,19). Collectively, these results were interpreted to suggest that rapid proton uptake to the D pathway (and proton pumping) cannot be maintained if Asp-132 is removed without introduction of a compensatory negative charge in the vicinity.…”

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase

“…The decoupling mutants maintain the O 2 reduction reaction [often at the same rate as the wild-type (WT) CcO], but fail to couple the chemical reaction to proton pumping (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Mutagenesis studies have further revealed several residues in the D-channel that are essential for efficient proton pumping.…”

“…S1A). It is the mutation of N139 (into various amino acid alternatives) that decouples proton pumping from chemical reaction most often (9)(10)(11)(14)(15)(16)(17)(18). However, how the mutation of N139 influences the key pumping and chemical reaction PT steps, which occur above E286 more than 25 Å away ( Fig.…”