Kinetic Control of O₂ Reactivity in H-NOX Domains

Abstract

Transient absorption, resonance Raman, and vibrational coherence spectroscopies are used to investigate the mechanisms of NO and O2 binding to WT Tt H-NOX and its P115A mutant. Vibrational coherence spectra of the oxy complexes provide clear evidence for the enhancement of an iron-histidine mode near 217 cm(-1) following photoexcitation, which indicates that O2 can be dissociated in these proteins. However, the quantum yield of O2 photolysis is low, particularly in the wild type (≲3%). Geminate recombination of O2 and NO in both of these proteins is very fast (∼1.4 × 10(11) s(-1)) and highly efficient. We show that the distal heme pocket of the H-NOX system forms an efficient trap that limits the O2 off-rate and determines the overall affinity. The distal pocket hydrogen bond, which appears to be stronger in the P115A mutant, may help retard the O2 ligand from escaping into the solvent following either photoinduced or thermal dissociation. This, along with a strengthening of the Fe-O2 bond that is correlated with the significant heme ruffing and saddling distortions, explains the unusually high O2 affinity of WT Tt H-NOX and the even higher affinity found in the P115A mutant.

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).…”

“…As a result, the authors reasoned that the absence of the proline residue causes an increase in the affinity for dioxygen, leading to a more stable Fe(II)-O 2 complex. This was elaborated in vibrational correlation spectroscopy experiments (Sun et al, 2016). Photolysis of the Fe–O 2 bond in wild-type and P115A Cs -H-NOX constructs revealed that the wild-type Fe–O 2 bond is more resistant to photolysis and has a longer O 2 -rebinding time in comparison to the P115A mutant.…”

“…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).…”

Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase

“…PCC7120 ( Ns H-NOX) [7,8,9,10]. On the other hand, H-NOXs found in obligate anaerobes are domains of methyl-accepting chemotaxis proteins (MCP) and often bind O 2 to form an oxyferrous complex, such as the ones from firmicutes Clostridium botulinum ( Cb H-NOX) and thermophilic Thermoanaerobacter tengcongensis ( Tt H-NOX, renamed Caldanaerobacter subterraneus H-NOX recently) [7,11,12,13]. …”