Stark-Effect Spectroscopy of the Heme Charge-Transfer Bands of Deoxymyoglobin

Abstract

We report the Stark spectra of the Soret, Q, and charge-transfer bands of deoxymyoglobin. The data show that band III has charge-transfer character but that the magnitude of the charge displacement is significantly smaller than expected or than what is observed for band I or even the Soret or Q bands. The data also show that symmetry breaking of iron-protoporphyrin IX in myoglobin produces a larger difference dipole moment in the Soret transition when compared to the dimethyl ester of protoporphyrin IX. The results lend further credence to the hypothesis that the electrostatic environment of the heme pocket in myoglobin is important in modifying the properties of the heme transitions, and they provide a basis for quantitative analysis of the static and time-dependent band shifts observed in response to environmental electrostatic perturbations and ligand binding dynamics.The electronic absorption of hemes has been investigated for more than 100 years. Ligand and substituent effects have been systematically assigned, and the focus in recent years has shifted toward static and dynamic band shifts that characterize biological function. [1][2][3][4][5][6] The origins of these band shifts can be roughly divided into specific chemical effects, e.g., changes in ligand or heme substituents, and more global environmental shifts, typically originating in changes in the electrostatic field around the chromophore. The latter effects are Stark shifts due to the interaction between the change in dipole moment (∆µ) and polarizability (∆R) of the chromophore with the electrostatic field of the ordered environment. To understand these electrostatic shifts quantitatively, it is necessary to characterize ∆µ and ∆R for each type of transition, and this can be achieved by measuring the effect of an externally applied electric field, the Stark-effect spectrum, for each transition. 7,8 The absorption spectra of heme and other metalloporphyrins are dominated by the B (Soret) and Q absorption bands that arise from configuration interaction (CI) of four orbitals, the nearly degenerate pair a 1u , a 2u HOMO and the doubly degenerate e 1g LUMO. 9,10 The result of CI is an intense Soret transition (λ max ≈ 435 nm in deoxymyoglobin, Mb) and a weak Q band (λ max ≈ 556 nm) that gains most of its intensity through vibronic coupling with the Soret band. 11 Although these bands dominate the spectra, there are other less prominent bands which are of interest. These include the four ligand-to-metal charge transfer (CT) and d-d electronic absorption transitions (bands I-IV) which have been assigned in the near-IR region of deoxyhemoglobin based on single-crystal absorption, CD, and MCD spectroscopy. 12 Band III is relatively narrow and easily resolved, and it has been shown that there are interesting correlations between the band position and ligand rebinding kinetics at cryogenic temperature in Mb. [1][2][3]13 This band exhibits timedependent spectral shifts after MbCO photolysis, and this has been taken to be an indicator of protein conformational...

CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier

“…9,31,[53][54][55][56][57][58][59][60][61][62] This band shifts to 770 nm in the absence of 2-MeIm as indicative of four-coordinate hemes. 35,63 Another band near 840 nm also appears when the imidazole ligand is absent and sharpens at low temperature (not shown).…”

“…31,56,57,62,64 On the other hand, it should be recognized that local electric fields can also influence the band III frequency. 59,60 Recently, a strong correlation between the Fe displacement and the oscillator strength of band III has been used 61 to explain the unusually strong temperature dependence observed 31 for band III in myoglobin. The proposed correlations 61 suggest that the blue shift and intensity decrease of band III, which are observed when 2-MeIm binds and displaces the water ligand to Fe(PPIX), reflect an increased Fe displacement from the heme plane and thus a higher barrier to CO binding.…”

Construção e estudos de perfomance de um reator fotoquímico tipo CPC ("Compound Parabolic Concentrator")

“…Esses termos são designações espectroscópicas para transições eletrônicas envolvendo orbitais moleculares π e π*. A banda Q é uma típica banda conjugada (ou de conjunto) e sua característica é uma intensa absortividade molar no visível [32][33][34][35] . Deve-se observar que a capacidade de aproveitamento de fótons para o processo catalítico por esses compósitos não se restringe a comprimentos de onda menores que 390 nm, como ocorre com o TiO 2 .…”

Blue protein with red fluorescence

“…This effect is called the Stark effect (47)(48)(49)(50). To elucidate if the Stark effect plays a role in the cause of redshift in the fluorescence of BLA on binding to Sandercyanin, we calculated the electrostatic potential of the Sandercyanin structure using the Adaptive Poisson-Boltzmann Solver.…”