Tracing the D-Pathway in Reconstituted Site-Directed Mutants of CytochromecOxidase fromParacoccus denitrificans

Abstract

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.

Replacing Asn207 by Aspartate at the Neck of the D Channel in the aa3-Type Cytochrome c Oxidase from Rhodobacter sphaeroides Results in Decoupling the Proton Pump

“…A similar mutation has also been reported at the equivalent position (N131D) in the oxidase from Paracoccus denitrificans (10). The N139D mutant of theR.…”

“…The question being addressed in the current work is whether mutating N207 to aspartate has a similar effect on the enzyme as does the N139N mutation. It has been shown that the equivalent mutation in the oxidase from P. denitrificans (N199D) results in decoupling the proton pump (10), but no additional characterization of this mutant has been reported. It is confirmed in the current work that the N207D mutant oxidase from R. sphaeroides also does not pump protons, similar to the behavior of N139D.…”

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3fromThermus thermophilus

“…Mutagenesis experiments have been performed on the equivalent residues: D399 in the P. denitrificans aa 3 -type oxygen reductase, D407 in the R. sphaeroides aa 3 -type oxygen reductase, and D407 in the Escherichia coli bo 3 -type oxygen reductase (14,27,(43)(44)(45)(46). The D399N mutation in the P. denitrificans oxidase does not significantly change the properties of the oxidase, whereas the D399L mutant has only ∼7% activity and does not pump protons (41). These data are similar to those reported in the current work with the T. thermophilus enzyme.…”

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

“…They appear to form a continuous network through hydrogen bonding between waters and with hydrophilic side chains, broken only by a conserved asparagine pair that forms a bridge interrupting the chain toward the entrance (13,(16)(17)(18). Several hydrophilic residues in the D pathway are crucial for the function of proton uptake, as evidenced by the effects of their mutation on enzyme activity and proton pumping efficiency (16,(19)(20)(21), in some cases inhibiting and others uncoupling activity.…”