Non-planar heme distortion in H-NOX

The H-NOX family of NO sensing proteins has received considerable attention because its members include the mammalian NO sensor, soluble guanylate cyclase (sGC). Despite this attention, the mechanism of signal transduction has not been elucidated. When NO binds sGC at the ferrous histidine-ligated protoporphyrin-IX, the proximal histidine ligand dissociates, resulting in a 5-coordinate (5c) complex; formation of this 5c complex is viewed as necessary for activation of sGC. Characterization of other H-NOX family members has revealed that while most also bind NO in a 5c complex, some bind NO in a 6-coordinate (6c) complex or as a 5c/6c mixture.

To gain insight into the heme pocket structural differences between 5c and 6c Fe(II)–NO H-NOX complexes, we investigated the Extended X-ray Absorption Fine Structure (EXAFS) of the Fe(II)–unligated and Fe(II)–NO complexes of H-NOX domains from several bacterial species with varyingFe(II)–NO coordination numbers. Independent of coordination number, we found that for all NO-bound H-NOX complexes, the EXAFS data are consistent with a weakly associated histidine. This study reveals that the overall heme structure of 5c and 6c Fe(II)–NO H-NOX complexes are substantially similar, suggesting that formal histidine dissociation is not required to trigger NO/H-NOX signal transduction. These results therefore suggest a mechanism for ready recycling of the enzymatic process, which would be needed for rapid response to fluctuating NO concentrations. An elongation, rather than rupture, of the Fe-His bond would retain the integrity of the active site and allow facile return to the resting state.

If dissociation of the proximal histidine bond is not required for signal transduction in the H-NOX family, what is the necessary change that occurs upon NO binding to initiate signal transduction? Structural studies of bacterial members of the family have revealed that the H-NOX heme cofactor is extremely distorted from planarity. Furthermore, it has been determined that heme distortion is maintained primarily by a conserved proline residue located in the proximal heme pocket. It has been suggested that changes in heme planarity may contribute to signal transduction. We have found that heme flattening is, indeed, sufficient for signal transduction in the H-NOX family. We demonstrated that mutation of the conserved proline to alanine, which results in heme flattening, has the same affect on regulation of downstream enzymatic processes as NO binding to wild-type H-NOX(we used NO/H-NOX regulation of c-di-GMP concentrations describedhereas the functional assay). This study demonstrates, for the first time, that heme flattening mimics the activated, NO-bound, state of H-NOX and suggests that NO binding induces heme flattening as part of the signal transduction mechanism in the H-NOX family.

Our model for signal transduction in the H-NOX family. Upon NO binding, the bond between the heme iron and the proximal histidine is lengthened, leading to an opening of the proximal pocket and effective removal of the heme-distorting proline residue. This results in heme flattening, which triggers an overall change in protein conformation and downstream signal transduction.

We are currently continuing our spectroscopic studies to explore the role of heme structure and protein environment in fine-tuning the electronic structure of the H-NOX heme. Furthermore, we are investigating how the electronic structure of heme contributes to the biological signaling function of H-NOX. Furthermore, through our basic, fundamental research into the biophysical and bioiorganic chemistry of H-NOX domains, we have also discovered new applications for H-NOX, including as a sensitive and practical cyanide sensor.

For more information see:

  • Sun, Y.; Benabbas, A.; Zeng, W.; Muralidharan, S.; Boon, E.M., Champion, P.M. (2016) Kinetic control of O2reactivity in H-NOX domains.Physical Chemistry B, 120, 5351-5358 (DOI:10.1021/acs.jpcb.6b03348).
  • Kosowicz, J.G.; Boon, E.M.* (2013) Insights into the distal pocket of H-NOX using fluoride as a probe for H-bonding interactions. Journal of Inorganic Biochemistry, 126, 91-95 (DOI: 10.1016/j.jinorgbio.2013.05.012).
  • Dai, Z.; Farquhar, E.R.; Arora, D.P.; Boon, E.M.* (2012) Is histidine dissociation a critical component of the NO/H-NOX signaling mechanism? Insights from X-ray absorbance spectroscopy. Dalton Transactions, 41, 7984-7993 (DOI: 10.1039/C2DT30147D).
  • Muralidharan, S.; Boon, E.M.* (2012) Heme flattening is sufficient for signal transduction in the H-NOX family. Journal of the American Chemical Society, 134, 2044-2046 (DOI: 10.1021/ja211576b).
  • Dai, Z.; Boon, E.M.* (2011) Probing the local electronic and geometric properties of the heme iron center in an O2-binding Heme-Nitric oxide and/or Oxygen binding domain. Journal of Inorganic Biochemistry, 105, 784-792 (DOI: 10.1016/j.jinorgbio.2011.03.002).
  • Dai, Z.; Boon, E.M.* (2010) Sensitive and selective detection of cyanide using an H-NOX domain. Journal of the American Chemical Society, 132, 11496-11503 (DOI: 10.1021/ja101674z).
Elizabeth Boon 2012