Complex Interactions of Carbon Monoxide with Reduced Cytochrome cbb 3 Oxidase from Pseudomonas stutzeri

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K C -channel is a conserved glutamate in subunit III. However, the majority of the K C -channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K Cchannel, was found to depend on the conformation of Y241 Vc , located in subunit I at the interface with subunit III. Mutations of Y241 Vc (to A/F/H/S) in the Vibrio cholerae cbb 3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b 3 . The data suggest a linkage between residues lining the K C -channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241 Vc and the active site crosslinked Y255 Vc , as well as two Cu B histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.oxygen reductase | proton pathway | bioenergetics | Vibrio cholerae | cbb 3

Section: Resultsmentioning

confidence: 62%

Conformational coupling between the active site and residues within the K ^C -channel of the Vibrio cholerae cbb ₃ -type (C-family) oxygen reductase

Ahn¹,

Mahinthichaichan²,

Lee³

et al. 2014

“…S3. After flash photolysis, the solubilized wild-type cbb 3 shows CO rebinding both to heme b 3 and to one of the c-type hemes (15,27), where the rapid phase represents the recombination to the c-type heme and the slow phase represents the b 3 -CO recombination. The E25 P -A and -Q variants show essentially the same rates and relative amplitudes of these phases, indicating that CO binding is unchanged in these mutants.…”

Section: Resultsmentioning

confidence: 99%

“…In cbb 3 , the complex behavior of CO binding (15,27) as well as the overlapping spectra of the three c-type hemes (see Figs. S2 and S3) have hampered detailed kinetic studies using the flowflash technique.…”

mentioning

confidence: 99%

Entrance of the proton pathway in cbb ₃ -type heme-copper oxidases

Lee

Gennis²,

Ädelroth

2011

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O 2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb 3 ) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb 3 numbering) in the accessory subunit CcoP (E25 P ). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb 3 by O 2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25 P as the entry point to the proton pathway in cbb 3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O 2 reduction. The cbb 3 is in addition to O 2 reduction capable of NO reduction, an activity that was largely retained in the E25 P variants, consistent with a scenario where NO reduction in cbb 3 uses protons from the periplasmic side of the membrane.glutamate | nitric oxide | oxygen reduction

“…The CO-bound protein (we noted that CO bound to both the b 3 and to one of the c-type hemes as described in ref. 32, but to a degree that varied between samples and did not significantly influence the results described) was then mixed with saturated O 2 (1.2 mM) or NO (2 mM) in 25 mM Hepes, 100 mM KCl (plus 0.01% DDM for solubilized protein) in a modified stopped-flow apparatus (Applied Photophysics). The protein concentration after mixing was 1.5-2 M. After a 200-ms delay, a laser flash (Nd:YAG laser, Quantel) was applied, dissociating CO from the active site and allowing the substrates to bind and oxidize the protein.…”

Section: Flow-flash Measurements; Optical Detection Of Heme Oxidationmentioning

confidence: 99%

Vectorial proton transfer coupled to reduction of O ₂ and NO by a heme-copper oxidase

Huang

Reimann

Lepp

et al. 2008

The heme-copper oxidase (HCuO) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. The HCuOs that reduce O 2 to H2O couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. The bacterial NO-reductases (NOR) reduce NO to N2O (2NO ؉ 2e ؊ ؉ 2H ؉ 3 N2O ؉ H2O), a reaction as exergonic as that with O2. Yet, in NOR both electrons and protons are taken from the outside periplasmic solution, thus not conserving the free energy available. The cbb 3-type HCuOs catalyze reduction of both O2 and NO. Here, we have investigated energy conservation in the Rhodobacter sphaeroides cbb3 oxidase during reduction of either O2 or NO. Whereas O2 reduction is coupled to buildup of a substantial electrochemical gradient across the membrane, NO reduction is not. This means that although the cbb3 oxidase has all of the structural elements for uptake of substrate protons from the inside, as well as for proton pumping, during NO reduction no pumping occurs and we suggest a scenario where substrate protons are derived from the outside solution. This would occur by a reversal of the proton pathway normally used for release of pumped protons. The consequences of our results for the general pumping mechanism in all HCuOs are discussed.cbb3 ͉ flow-flash ͉ nitric oxide ͉ proton pumping ͉ pathway