Identification of the overtone of the Fe-CO stretching mode in heme proteins: a probe of the heme active site.

Abstract

Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000-to 1070-cm'1 region. The other line is found in the 1180-to 1210-cmn-' region and is assigned as a combination between a porphyrin mode, V7, and the F6-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-0 stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.Heme proteins, which function either as oxygen carriers or as catalytic enzymes, have a pocket on the distal side of the heme in which exogenous ligands bind to the iron atom. The function of the heme protein depends critically on the properties of the distal pocket and how its residues interact with the hemebound exogenous ligands. Studies of CO adducts of heme proteins have been shown to be extremely important probes of the active sites, including the interactions between the CO and the distal residues (1, 2). In addition, CO photodissociation and recombination studies have played an essential role in unraveling the dynamic processes that occur in heme proteins, information which has led to an advanced understanding of the basic physics that forms the foundation of the physiological process of ligand binding (3, 4).Structural information such as the identity of the proximal ligand, the geometry of the bound CO, and the interactions between the axial ligands and the heme environment has been determined from the vibrational spectra of the fundamental modes involving the CO. However, overtones offer the possibility of yielding additional information concerning the heme pocket and the electronic structure because the behavior of the overtones depends critically on the shape of the ground-state potential function as well as the properties of the excited electronic state. In the overtone region of the Fe-CO stretching mode ("Feco) two lines have been independently discovered recently (5, 6). We assign one as the overtone ("over) Of the Fe-CO stretching mode and the second as a combination (Vcomb) of the Fe-CO stretching mode with a mode of the porphyrin macrocycle (V7...

The Role of the Cross-link His-Tyr in the Functional Properties of the Binuclear Center in Cytochrome c Oxidase

“…The modes at 1464 and 1490 cm Ϫ1 originate from the ferrous, five-coordinate high spin heme a 3 2ϩ and the ferrous six-coordinate low-spin heme a, respectively. The mode at 1519 originates from 19 of heme a 2ϩ . The modes at 1569 and 1584 cm Ϫ1 originate from 2 of heme a 3 2ϩ and heme a, respectively.…”

Red Blood Cells Polarize Green Laser Light Revealing Hemoglobin′s Enhanced Non‐Fundamental Raman Modes

“…Non-FC dependence of the electronic transition moment on the vibrational coordinate is possible through B-term and C-term enhancement. [6] When the excitation is in resonance with the weaker Q 0 band at approximately 575 nm, B-term scattering dominates,[7] which involves vibronic coupling between two allowed excited electronic transitions. Early studies on metalloporphyrins in solution showed that both Q 0 and Q v bands are predominantly governed by the strong Herzberg–Teller coupling of antisymmetric A 2g modes,[8, 9] providing evidence of vibronic coupling.…”

“…[7, 17] Strekas and Spiro reported that, although RR spectra sometimes display intense overtone progressions, these are not apparent in the RR spectra of Hb. [20] It is important to note that these early reports were based on what was known from solution-phase studies.…”

“…Figureshowsthe allowed electronic transitions based on the Gouterman four-orbitalmodel along with the laser lines used in this experiment (color coded)and the symmetry terms of the bands that are enhanced at the threemajor electronic transitions. mately 575 nm, B-term scattering dominates, [7] which involves vibronic coupling between two allowed excited electronic transitions. Early studies on metalloporphyrins in solution showed that both Q 0 and Q v bands are predominantly governed by the strong Herzberg-Teller coupling of antisymmetricA 2g modes, [8,9] providing evidenceo fv ibronic coupling.…”

“…[17] It was furtherp ostulated that heme proteins such as Hb fall into the category of weak coupling compounds,b ecause the electronic transitions of these compounds are distributed over severaln uclei. [7,17] Strekasa nd Spiro reported that, although RR spectra sometimesd isplay intense overtone progressions, these are not apparent in the RR spectra of Hb. [20] It is important to note that these early reports were based on what was known from solution-phase studies.…”