Engineering proximal vs. distal heme-NO coordination via dinitrosyl dynamics: implications for NO sensor design.

Proximal vs. distal heme-NO coordination is a novel strategy for selective gas response in heme-based NO-sensors. In the case of Alcaligenes xylosoxidans cytochrome c' (AXCP), formation of a transient distal 6cNO complex is followed by scission of the trans Fe-His bond and conversion to a proximal 5cNO product via a putative dinitrosyl species. Here we show that replacement of the AXCP distal Leu16 residue with smaller or similar sized residues (Ala, Val or Ile) traps the distal 6cNO complex, whereas Leu or Phe residues lead to a proximal 5cNO product with a transient or non-detectable distal 6cNO precursor. Crystallographic, spectroscopic, and kinetic measurements of 6cNO AXCP complexes show that increased distal steric hindrance leads to distortion of the Fe-N-O angle and flipping of the heme 7-propionate. However, it is the kinetic parameters of the distal NO ligand that determine whether 6cNO or proximal 5cNO end products are formed. Our data support a 'balance of affinities' mechanism in which proximal 5cNO coordination depends on relatively rapid release of the distal NO from the dinitrosyl precursor. This mechanism, which is applicable to other proteins that form transient dinitrosyls, represents a novel strategy for 5cNO formation that does not rely on an inherently weak Fe-His bond. Our data suggest a general means of engineering selective gas response into biologically-derived gas sensors in synthetic biology.

A distal pocket Leu residue inhibits the binding of O2 and NO at the distal heme site of cytochrome c'.

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off). The connection between distal steric constraints, k(off) values, and distal to proximal heme-NO conversion is discussed.

Activation parameters for heme-NO binding in alcaligenes xylosoxidans cytochrome c': the putative dinitrosyl intermediate forms via a dissociative mechanism.

The bacterial heme protein Alcaligenes xylosoxidans cytochrome c' (AXCP) forms a novel five-coordinate heme-nitrosyl (5c-NO) complex in which NO resides at the proximal heme face in place of the endogenous protein ligand. Intriguingly, AXCP shares NO-binding properties with the eukaryotic NO-sensor, soluble guanylate cyclase (sGC), including 5c-NO formation via two NO-dependent reactions. For both proteins, a model has been proposed in which NO binds to the vacant distal face to form a transient six-coordinate heme-nitrosyl (6c-NO) species, which then converts to a proximal 5c-NO complex via a putative dinitrosyl intermediate. To shed light on this novel reaction mechanism, activation parameters have been determined for distal and proximal NO-binding reactions in AXCP from the effect of temperature and hydrostatic pressure on rate constants. The unusually slow 6c-NO formation reaction has a near-zero entropy of activation and a positive volume of activation (DeltaV(double dagger) = +14.1 cm(3) mol(-1)), consistent with a rate-determining step involving movement of the Leu 16 residue to allow NO binding to the crowded distal site. For the 6c-NO --> 5c-NO conversion, the large positive entropy of activation (DeltaS(double dagger) = +103 J K(-1) mol(-1)) and volume of activation (DeltaV(double dagger) = +24.1 cm(3) mol(-1)) suggest that the putative dinitrosyl intermediate forms via a dissociative mechanism in which the endogenous His ligand dissociates prior to the attack of the second NO molecule on the proximal heme face. These results have important implications for distal vs proximal NO binding in AXCP, as well as mechanisms of 5c-NO formation in heme proteins.