Activation Parameters for Heme−NO Binding in Alcaligenes xylosoxidans Cytochrome c′: The Putative Dinitrosyl Intermediate Forms via a Dissociative Mechanism

• Published in: Journal of the American Chemical Society 2009-04, Vol.131 (13), p.4846-4853
• Main Authors: Pixton, David A, Petersen, Christine A, Franke, Alicja, van Eldik, Rudi, Garton, Elizabeth M, Andrew, Colin R
• Format: Article
• Language: English
• Subjects: Alcaligenes - enzymology; Bacterial Proteins - metabolism; Binding Sites; Cytochromes c' - metabolism; Heme - metabolism; Kinetics; Ligands; Nitric Oxide - metabolism; Thermodynamics
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja809587q

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 (ΔV ⧧ = +14.1 cm3 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 (ΔS ⧧ = +103 J K−1 mol−1) and volume of activation (ΔV ⧧ = +24.1 cm3 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.