Looking for the minimum common denominator in haem–copper oxygen reductases: Towards a unified catalytic mechanism

102

185

The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B-and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D-and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba 3 oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O 2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidinetyrosine, the Cu B and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.cytochrome c oxidase ͉ respiratory enzymes

Section: Discussionmentioning

confidence: 99%

The cytochrome ba ₃ oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

Chang¹,

Hemp

Chen

et al. 2009

102

185

“…T he oxygen reductase members of the heme-copper superfamily are critical redox-driven proton pumps in the aerobic respiratory chains of most aerobic bacteria and archaea as well as in the mitochondria of eukaryotes (1)(2)(3)(4). These membranebound proteins catalyze the reduction of O 2 to water and use the free energy of this reaction to generate a proton motive force that is used for a number of essential biological functions, including the synthesis of ATP.…”

mentioning

confidence: 99%

“…The heme-copper oxygen reductases constitute a diverse group of phylogenetically related enzymes, the vast majority of which can be classified as members of the A-, B-, or C-family of hemecopper oxygen reductases (1,5,6). Enzymes from each of these oxygen reductase families have been shown to couple the redox chemistry to proton pumping (7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

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

Chang¹,

Choi

Vakkasoglu

et al. 2012

The heme-copper oxygen reductases are redox-driven proton pumps. In the current work, the effects of mutations in a proposed exit pathway for pumped protons are examined in the ba 3 -type oxygen reductase from Thermus thermophilus , leading from the propionates of heme a 3 to the interface between subunits I and II. Recent studies have proposed important roles for His376 and Asp372, both of which are hydrogen-bonded to propionate-A of heme a 3 , and for Glu126 II (subunit II), which is hydrogen-bonded to His376. Based on the current results, His376, Glu126 II , and Asp372 are not essential for either oxidase activity or proton pumping. In addition, Tyr133, which is hydrogen-bonded to propionate-D of heme a 3 , was also shown not to be essential for function. However, two mutations of the residues hydrogen-bonded to propionate-A, Asp372Ile and His376Asn, retain high electron transfer activity and normal spectral features but, in different preparations, either do not pump protons or exhibit substantially diminished proton pumping. It is concluded that either propionate-A of heme a 3 or possibly the cluster of groups centered about the conserved water molecule that hydrogen-bonds to both propionates-A and -D of heme a 3 is a good candidate to be the proton loading site.

“…The active sites of both NOR and HCO contain a proximal histidine-coordinated heme and a distal three histidine-coordinated metal center. However, the metal center in HCOs is occupied by a copper (called Cu B ), whereas a nonheme iron is present in NOR (called Fe B ) (23,24). In addition, two conserved glutamates, shown by modeling to be close to the Fe B site (21, 22), are found to be essential for NOR activity (24,25).…”

mentioning

confidence: 99%

“…Bacterial NOR is a membrane-bound protein that catalyzes one step of this process, namely, the two-electron reduction of NO to N 2 O (17,19). With no crystal or solution structure available for bacterial NOR to date, sequence alignments and homology modeling (21,22) have indicated that NOR is structurally homologous to the largest subunit (subunit I) of hemecopper oxidases (HCOs) (23), enzymes that catalyze reduction of O 2 to water. The active sites of both NOR and HCO contain a proximal histidine-coordinated heme and a distal three histidine-coordinated metal center.…”

mentioning

confidence: 99%

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

Lin

Yeung²,

Gao³

et al. 2010

108

140

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe B site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by ∼110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ∼100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe B site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more welldefined system to address important chemical and biological issues.biomimetic models | heme-copper oxidase | metalloprotein | protein design | protein engineering R ational design of proteins that mimic both structure and function of more complex native enzymes has been a long soughtafter goal, as the process is an ultimate test of our knowledge and an excellent means to develop advanced biocatalysts (1-3). Although designed proteins that model the structure of native enzymes have been known for a while (4-10), successful designs of proteins that mimic both the structure and function of native enzymes have been reported only recently (11-16). While being able to design such functional proteins is laudable, the impact of such an achievement would be greater if the designed proteins can be used to address fundamental issues in chemistry and biology that are difficult to tackle by other methods. One primary example is the roles of conserved glutamates and metal ions in bacterial nitric oxide reductase (NOR) (17)(18)(19).NO is critical for all life (20). Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17, 19). Bacterial NOR is a membrane-bound protein that catalyzes one step of this process, namely, the two-electron reduction of NO to N 2 O (17, 19). With no crystal or solution structure available for bacterial NOR to date, sequence alignments and homology modeling (21, 22) have indicated that NOR is structurally homologous to the largest subunit (subunit I) of hemecopper oxidases (HCOs) (23), enzymes that catalyze reduction of O 2 to water. The active sites of both NOR and HCO contain a proximal histidine-coo...