Structure

Overall structures and active site structures

qNOR structure viewed parallel to the membrane and the arrangement of the qNOR membrane-spanning helices viewed from the cytoplasmic side.

• As the figure shows, G.stearothermophilus qNOR contains a membrane-spanning region consisting of 14 transmembrane helices and α-helical hydrophilic domain on the extracellular side. 
• comparing the overall structure of the P.aeruginosa cNOR and qNOR: 

Common features:

• A calcium ion bridges the heme propionates from heme b and b3 in both enzymes
• Overall structure of C terminus of qNOR is similar to the norB subunit of cNor. 
• Except α3 helix, 4 of the 5 alpha-helices in qNOR hydrophilic domain show substantial structural homology both with Cyt-c domain of the NorC subunit in cNOR and with the Cyt--c551 protein.

Differences: The qNOR hydrophilic domain contains neither heme c nor its binding motif Cys-X-X-Cys-His

The removal of heme from holocytochrome-c causes loss of the native folding pattern, but in qNOR, several bulky hydrophobic residues compensate for the loss of the hydrophobic heme macrocycle and maintain Cyt-c folding.(left)

Heme c of the superimposed cyt-c₅₅₁ is shown as a red stick, the bulky hydrophobic residues in qNOR around the overlaid heme c are shown as blue sticks. (right)

The transmembrane domain portion of qNOR is characterized by low-spin heme b, high-spin heme b3 and non-heme metal. In addition, one calcium ion was found between heme b and b3.

The conserved His355 and His653 residues serve as the ligands for heme b, and the conserved His651 residue is the ligand for heme b3.

The binuclear center of qNOR is formed by the non-heme metal and the heme b3 iron, which are 4.6 Å apart.

a) redox centres of qNOR, b) environment of calcium in qNOR c)structure of binuclear centre of qNOR d) structure of binuclear centre in cNOR

http://www.nature.com/nsmb/journal/v19/n2/full/nsmb.2213.html

Zinc can also be found at the non-heme metal site (ZnB). Three histidine residues(His 508, His 509 and His560) are ligands for the ZnB, while it is also coordinated by two water molecules. Thus ZnB adopts a five-coordinate structure with distorted  trigonal bipyramidal geometry. Glu512 does not coordinate to the ZnB because it is 5.4Å away.

In contrast, cNOR (figure.d) uses iron as its non heme metal, coordinated by three histidine and one glutamate Glu211. 

Proton transfer

It has been suggested that both Glu512 and Glu581 (located close to the binuclear centre are involved in the proton transfer for catalytic NO reduction.

qNOR

• The qNOR mutants E512A and E581A presented no catalytic activity, suggesting that these two conserved glutamate residues are essential for NO reduction.
• There is a water cluster present around the heme b and b3 propionates. There is neither an obvious water channel nor a hydrogen bond network connecting these glutamate residues to the extracellular surface.
• Instead there is a hydrophilic channel, which contains many ordered waters and is lined with polar residues, and extends from the binuclear centre to the cytoplasm. 

      The diagram below shows a schematic representation of proton transfer (top) and crystal structure of qNOR with the hydrophilic channel shown (bottom)

http://www.riken.jp/engn/r-world/info/release/press/2012/120123/index.html

 The paper proposes that the hydrophilic channel (water channel) can serve as a pathway for the catalytic protons used in the NO reduction at the binuclear centre.

Pathway of the protons

•  Protons come into the channel through the salt bridge.
• Transferred to chain of water molecules in the water channel

•   Hydrogen bonds between the water molecules are forming and breaking on a 100-ps time scale, this can efficiently transfer the protons.

-          Protons donated to the binuclear centre through the Glu512 and Glu581 (potential terminal proton donors).

Dynamics in the water channel of qNOR

• Side chain fluctuations
• Moving water molecules
• Forming and breaking of transient hydrogen bond networks.
  

Water molecules in the channel 

 The hydrophilic channel is shown in the video to the above in qNOR

• They are fairly mobile
• Water molecules entering the pore form the bulk water exchange with the water molecules inside the channel, and then having spent some time there return to the bulk water. 
•    (Glu281 – from salt bridge, can assist bulk water in entering the water channel)

This supports the suggestion that the hydrophilic channel acts as a proton transfer pathway for the catalytic reaction.

Electron transfer

• The electrons used in NO reduction by qNOR are supplied from menaquinol to the binuclear center through heme b.  Electron density in the F_o – F_c omit map indicated  that HQNO binds to the enzyme surface 10 Å away from the heme b iron and close to the headgroup region of the membrane's extracellular side.
  
  
  The first example of a structurally determined quinol binding site has been demonstrated. The quinol analogue HQNO is shown in pink.
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  The OH group of quinol analog HQNO forms a hydrogen bond with the carboxylate of Asp746, which is very commonly observed among quinol-binding enzymes. Binding also involves the N-oxide forming hydrogen bond with the imidazole ring of His328, which is connected to the extracellular surface by a hydrogen bond network through glu322.
•  the residues around the HQNO-binding site (Asp746, His328, Glu332, Phe336 and Phe337) are highly conserved in qNORs but not in cNORs.
  
  
  A short video shows the arrangement of quinol binding site and proximity to heme center.

Two electrons from menaquinol are donated to heme b₃ in the binuclear center through heme b. The presence of a calcium ion between the two hemes is crucial to the enzymatic function as When the calcium ligands Tyr93 or Glu429 were mutated, the calcium content and the enzymatic activity decreased.The calcium ion in qNOR may maintain the protein conformation, creating an efficient electron transfer from the heme b to the heme b₃ iron and regulating the redox potential of heme irons.