Probing the local electronic and geometric properties of the heme iron center in a H-NOX domain

Abstract

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.

Bacterial Haemoprotein Sensors of NO

“…Studies of the Pro115 mutant of Cs -H-NOX (haem-flattened mutant) have bolstered this hypothesis (Dai & Boon, 2011; Olea et al, 2008; Sun et al, 2016). The Cs -H-NOX P115A mutant was shown to have a significantly slower O 2 dissociation rate constant in comparison to wild-type Cs -H-NOX, but the O 2 association rate constant remained unchanged (Olea et al, 2008).…”

“…The structure of ferrous-oxy Cs -H-NOX also led to the identification of a unique structural feature of the H-NOX family: a highly distorted haem cofactor, as well as the suggestion that a proline residue [Pro115 in Cs -H-NOX; this proline is absolutely conserved in the H-NOX family (Karow et al, 2004)] is vitally important in maintaining this haem distortion (Dai & Boon, 2011; Erbil, Price, Wemmer, & Marletta, 2009; Olea, Boon, Pellicena, Kuriyan, & Marletta, 2008; Olea, Kuriyan, & Marletta, 2010; Sun et al, 2016). To assess the significance of Pro115 in haem distortion in Cs -H-NOX, the proline to alanine mutant was crystallized and solved to 2.1 Å (Olea et al, 2008).…”

“…Cs -H-NOX has also been studied by X-ray absorption near edge spectroscopy (XANES) (Dai & Boon, 2011). The excitation energy of the edge of the iron K-shell, the innermost electron shell, is useful for understanding the coordination, oxidation, and ligand-binding properties of haem iron.…”

“…Several key structural features have been identified in NO-activation of H-NOX, including the dissociation of a proximal histidine ligand (Dai et al, 2012; Erbil et al, 2009; Herzik et al, 2014; Olea, Herzik, et al, 2010) and flattening of a haem cofactor (Dai & Boon, 2011; Erbil et al, 2009; Olea et al, 2008; Olea, Kuriyan et al, 2010; Sun et al, 2016) that is otherwise exceedingly distorted. Features specific to some H-NOX proteins have also been distinguished, including a hydrogen-bonding network that stabilizes O 2 binding in H-NOX from obligate anaerobes (Boon et al, 2005; Hespen et al, 2016) and a zinc-binding feature that may be connected to redox sensing in H-NOX domains from some gammaproteobacteria (Mukhopadyay et al, 2016).…”

Porphyrin-Substituted H-NOX Proteins as High-Relaxivity MRI Contrast Agents

“…Relaxivities determined for high-spin, ferric aqua Mb (1.6 and 2.9 mM –1 s –1 for T 1 and T 2 , respectively) were similar to previously reported values. 16 The analogous Tt H-NOX high-spin ferric complex 17 displayed significantly improved T 1 and T 2 relaxivities of 5.9 and 14.5 mM –1 s –1 , respectively (Figure 1 and Table 1). Tt H-NOX is a larger protein (22 kDa vs. 17 kDa) with a more extended conformation, which could increase relaxivity by slowing solution tumbling.…”