Resonance Raman Spectra of Five-Coordinate Heme-Nitrosyl Cytochromes c': Effect of the Proximal Heme-NO Environment.

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

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.