Journal
JOURNAL OF INORGANIC BIOCHEMISTRY
Volume 105, Issue 6, Pages 784-792Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2011.03.002
Keywords
Neme-nitric oxide/oxygen binding domain; NO/sGC signaling; X-ray absorption near-edge structure spectroscopy; Edge; Ligand field indicator ratio; Iron-heme displacement
Funding
- New York State Foundation for Science, Technology and Information [C060030]
- NSF [CHE-0910771]
- Stony Brook University
- National Institute for Biomedical Imaging and Bioengineering [P30-EB-09998]
- U.S. DOE, Office of Science, Office of Basic Energy Sciences [DE-AC02-98CH10886]
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [0910771] Funding Source: National Science Foundation
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Heme-Nitric oxide and/or OXygen binding (H-NOX) proteins are a family of diatomic gas binding hemoproteins that have attracted intense research interest. Here we employ X-ray absorption near-edge structure (XANES) spectroscopy to study the nitric oxide (NO) binding site of H-NOX. This is the first time this technique has been utilized to examine the NO/H-NOX signaling pathway. XANES spectra of wildtype and a point mutant (proline 115 to alanine, P115A) of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) were obtained and analyzed for ferrous and ferric complexes of the protein. This work provides specific structural characterization of the solution state of several Tt H-NOX ferrous complexes (-unligated, -NO, and -CO) that were previously unavailable. Our iron K-edges indicate effective charge on the iron center in the various complexes and report on the electronic environment of heme iron. We analyzed the ligand field indicator ratio (LFIR), which is extracted from XANES spectra, for each complex, providing an understanding of ligand field strength, spin state of the central iron, movement of the iron atom upon ligation, and ligand binding properties. In particular, our LFIRs indicate that the heme iron is dramatically displaced towards the distal pocket during ligand binding. Based on these results, we propose that iron displacement towards the distal heme pocket is an essential step in signal initiation in H-NOX proteins. This provides a mechanistic link between ligand binding and the changes in heme and protein conformation that have been observed for H-NOX family members during signaling. (C) 2011 Elsevier Inc. All rights reserved.
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