Resonance Raman studies of cytochrome c' support the binding of NO and CO to opposite sides of the heme: implications for ligand discrimination in heme-based sensors

Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidans cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO and CO but not O(2). Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sides of its heme (distal and proximal) for binding exogenous ligands to its Fe center. The present RR investigation of the Fe coordination and heme pocket environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' in solution fully supports the structures determined by X-ray crystallography and offers insights into mechanisms of ligand discrimination in heme-based sensors. Ferrous cytochrome c' reacts with CO to form a six-coordinate heme-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively high stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond. RR spectra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal heme pocket. On the other hand, RR data for the five-coordinate NO adduct suggest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme. Parallels between certain physicochemical properties of cytochrome c' and those of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO binding.     

Heme displacement mechanism of CooA activation: mutational and Raman spectroscopic evidence

The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change.     

Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.