Heme proteins bind the gaseous ligands XO (X = C, N, O) via backbonding from Fe d π electrons. Backbonding is modulated by distal interactions of the bound ligand with the surrounding protein and by variations in the strength of the trans proximal ligand. Vibrational modes associated with FeX and XO bond stretching coordinates report on these interactions, but the interpretive framework developed for CO adducts, involving anticorrelations of νFeC and νCO, has seemed not to apply to NO adducts. We have now obtained an excellent anticorrelation of νFeN and νNO, via resonance Raman spectroscopy on (N-methylimidazole)Fe(II)TPP-Y(NO), where TPP-Y is tetraphenylporphine with electron donating or withdrawing substituents, Y, that modulate the backbonding; the problem of laser-induced dissociation of the axial base was circumvented by using frozen solutions. New data are also reported for CO adducts. The anticorrelations are supported by DFT calculations of structures and spectra. When protein data are examined, the NO adducts show large deviations from the modeled anticorrelation when there are distal H-bonds or positive charges. These deviations are proposed to result from closing of the FeNO angle due to a shift in the valence isomer equilibrium toward the Fe(III)(NO -) form, an effect that is absent in CO adducts. The differing vibrational patterns of CO and NO adducts provide complementary information with respect to protein interactions, which may help to elucidate the mechanisms of ligand discrimination and signaling in heme sensor proteins.

Determinants of the Fe-CO and C-O stretching frequencies in (imidazole) heme-CO adducts have been investigated via Density Functional Theory (DFT) analysis, in connection with puzzling characteristics of the heme sensor protein CooA, and of the H-NOX (Heme-Nitric Oxide and/or OXygen binding) family of proteins, including soluble guanylate cyclase (sGC). The computations show that two mechanisms of Fe-histidine bond weakening have opposite effects on the νFeC/νCO pattern. Mechanical tension is expected to raise νFeC with little change in νCO, while weakening of H-bond donation from the imidazole ligand has the opposite effect. Data on CooA indicate imidazole H-bond weakening associated with heme displacement, as part of the activation mechanism. The computations also reveal that protein-induced distortion of the porphyrin ring, a prominent structural feature of the H-NOX protein TtTar4H (Thermoanaerobacter tengcongensis Tar4 protein), has surprisingly little effect on νFeC or νCO. However, another structural feature, strong H-bonding to the propionates, is suggested to account for the weakened backbonding that is evident in sGC. TtTar4H-CO itself has an elevated νFeC, which is successfully modeled as a compression effect, resulting from steric crowding in the distal pocket. νFeC/νCO data, in conjunction with modeling, can provide valuable insight into mechanisms for heme-protein modulation.

Resonance Raman studies have uncovered puzzling complexities in the structures of NO adducts of heme proteins. Although CO adducts of heme proteins obey well-behaved anti-correlations between Fe-C and C-O stretching frequencies, which reflect changes in backbonding induced by distal H-bonding residues, the corresponding NO data are scattered. This scatter can be traced to distal influences, since protein-free NO-hemes do show well-behaved anti-correlations. Why do distal effects produce irregularities in vFeN/ vNO plots but not in vFeC/vCO plots? We show via density functional theory (DFT) computations on model systems that the response to distal H-bonding differs markedly when the NO acceptor atom is N versus O. Backbonding is augmented by H-bonding to O, but the effect of H-bonding to N is to weaken both N-O and N-Fe bonds. The resulting downward deviation from the vFeN/vNO backbonding line increases with increasing H-bond strength. This effect explains the deviations observed for a series of myoglobin variants, in which the strength of distal H-bonding is modulated by distal pocket residue substitutions. Most of the data follow a positive vFeN/vNO correlation with the same slope as that calculated for H-bonding to N. Such deviations are not observed for CO adducts, because the CO pi* orbital is unoccupied, and serves as a delocalized acceptor of H-bonds. H-bonding to N primes NO-heme for reduction to the HNO adduct, a putative intermediate in NO-reducing enzymes.

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme:substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UVvis) difference spectroscopic studies demonstrated that in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm −1 in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm −1 in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge-transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.