Protonation of porphyrin in iron-free cytochromec: Spectral properties of monocation free base porphyrin, a charge analogue of ferric heme

Abstract

Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal‐free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately α‐helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is ∼ 3 kcal. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 141–150, 1999

Structural dynamics and reactivity of a cationic mono(acridyl)bis(arginyl)porphyrin: A spectroscopic study down to femtoseconds

“…l exc ¼ 322 nm. 25 is quite similar to that of H 2 TMPyP. 19 During the observation time (5 ps), the differential absorption spectrum increases in the spectral range 440-480 nm and decreases between 480 and 530 nm without any important spectral deformation.…”

“…In contrast, only one band centred at 565 nm with a shoulder at 522 nm appears in the excitation spectra performed at excitation wavelengths of 620 and 630 nm. These excitation spectra are similar to the absorption spectra of symmetric tetrapyrrole rings such as metalloporphyrins 20 and fully protonated/deprotonated porphyrins 25 or of porphyrins which form hydrogen bonds. 26,27 Time-resolved fluorescence spectroscopy.…”

“…The absence of the P aH bands in the BAP and H 2 TMPyP spectra confirms this attribution. Although the absorption spectrum of P aH resembles those of monocationic porphyrins previously identified from cytochrome c 25 and protoporphyrin, 32 a proton transfer between porphyrin and acridine inside MABAP could not occur. Indeed, at pH 7.0, the acridine chromophore is deprotonated (pK a1 ¼ 5.6) 33 so that such a transfer may only occur from porphyrin to acridine leading to the formation of the acridinium ion.…”

“…Due to the folding of the MABAP molecule, one could expect the formation of a hydrogen bond between acridine and one of the hydrogen atoms of the pyrrole N-H groups of the porphyrin. 25,26 Indeed, it has been demonstrated that an important decrease of the acridine fluorescence lifetime can be related to the formation of a hydrogen bond implying the nitrogen atom of the chromophore. 31 This hypothesis is in agreement with the spectroscopic properties of the acridine chromophore.…”

Photodynamic inactivation of Escherichia coli by porphyrin cytochrome c

“…The phototoxicity of PPIX, a porphyrin that has a similar structure as the porphyrin inside iron free Cyt c (for their structures, see Fig. S5, ESIw), 24 was also assessed. As shown in Fig.…”