We have measured and analyzed the pH dependence of the 695 nm charge transfer band of horse heart ferricytochrome c as a function of pH between 7.0 and 10.5 at high (50 mM) and low (0.5 mM) phosphate ion concentrations. Our data clearly reveal that the transition from the native state (III) to the two alkaline states (IV) involves two deprotonation steps which cannot be assigned to the two different lysine ligands associated with the two alkaline states. While the respective pK values are rather similar at high phosphate concentrations (9.23 and 9.14), they are clearly different at low anion concentrations (9.65 and 8.5). Apparently, the deprotonation that can be assigned to a pK of 8.5 populates an intermediate state termed III*, in which M80 is still an axial ligand. A comparison of Soret band CD spectra suggests that III* bears some similarity with the recently characterized thermally excited state IIIh. Our data suggest that the current picture of the alkaline transition is incomplete. The obtained results might be of relevance for characterizing the structure of ferricytochrome c bound to anionic phospholipids.

The N- and C-terminal blocked hexapeptide Ac-Leu-Ala-His-Tyr-Asn-Lys-amide (LAHYNK) representing the 80–85 fragment of histone H2B was synthesized and its interactions with Cu(II) and Ni(II) ions were studied by potentiometric, UV-Vis, CD, EPR, and NMR spectroscopic techniques in solution. Our data reveal that the imidazole N(3) nitrogen atom is the primary ligating group for both metal ions. Sequential amide groups deprotonation and subsequent coordination to metal ions indicated an {Nimidazole, 3Namide} coordination mode above pH∼9, in all cases. In the case of Cu(II)-peptide system, the almost exclusive formation of the predominant species CuL in neutral media accounting for almost 98% of the total metal ion concentration at pH 7.3 strongly indicates that at physiological pH values the sequence -LAHYNK- of histone H2B provides very efficient binding sites for metal ions. The imidazole pyrrole N(1) ionization (but not coordination) was also detected in species CuH−4L present in solution above pH ∼ 11.

Complexes with bulky hydrotris(triazolyl)borate (Ttz) ligands, TtzCuCO, were used to probe how acids change the donor properties of Ttz ligands. (Ttz(tBu,Me))CuCO shows four distinct protonation states and a gradual increase in the CO stretch. The increased electrophilic nature of the Cu center upon protonation leads to enhanced C-H activation catalysis.

We determined the dispersion of the depolarization ratio (DPR) of several Raman bands of ferricytochrome c at different pH values using low-ionic-strength conditions. The experimental data predominantly cover the pre-resonance and resonance region of Q-band excitation. The selected pH values 7.5, 9.0, 10.0 and 11.0 correspond to the conformational states III, III * , IV, and V of the protein. While the existence of the states III, IV, and V is known for nearly 70 years, the nativelike state III * has only recently been obtained from optical measurements at low ionic strength [D. Verbaro, A. Hagarman, J. B. Soffer and R. Schweitzer-Stenner, Biochemistry, 48, 2990, 2009]. We used group-theoretical arguments to obtain in-plane symmetry-lowering deformations from the obtained DPRs of various Raman bands in the high-wavenumber region of the obtained spectra. Thus, we found that a comparatively strong rhombic deformation along the B 1g mode, ν 18 , is maintained at all pH values investigated. It most likely arises from static Jahn-Teller distortion of the E-symmetry ground state. While this distortion depends on the existence of a strong sixth ligand, its occurrence is rather independent of the nature of this ligand as long as a low-spin configuration is maintained. The III → III * transition was found to modify A 1g -type perturbations of the heme macrocycle. This is likely to reflect a decrease in nonplanar distortions such as ruffling and saddling. This drop in nonplanarity is slightly reversed by the subsequent transitions into states IV and V. Circular dichroism (CD) spectra of the Q-band region suggest that the III → III * transition reduces the electronic contribution to the Q-band splitting, which could reflect either a weakening or a reorientation of the internal electric field in the heme pocket. Our results underscore the relevance of state III * as a thermodynamic intermediate of the alkaline transition between states III and IV.

This paper reports the discovery of a (meta)stable partially unfolded state of horse heart ferricytochrome c that was obtained after exposing the protein to a solution with an alkaline pH of 11.5 for 1 week. Thereafter, the protein did not undergo any detectable change in its secondary and tertiary structure upon adjusting the solution to folding promoting conditions at neutral pH. Spectroscopic data suggest that the misfolded protein exhibits a hexacoordinated low-spin state with a hydroxyl ion as the likely ligand. Below pH 6, a new ligation state emerges with the spectroscopic characteristics of a pentacoordinated quantum mixed state of the heme iron. Gel electrophoresis revealed substantial formation of soluble dimers and trimers at submillimolar concentrations, whereas monomers were dominant at lower, micromolar concentrations. Ultraviolet circular dichroism spectra indicate that oxidized monomers are pre-molten globule to globule-like with a substantial fraction of secondary (helical) structure reminiscent of alkaline state V. The oligomers contain even more helical structure, which suggests domain swapping as the underlying mechanism of their formation. A substantial fraction of the submillimolar mixture of monomers and oligomers underwent a reduction of the heme iron. Its dependence on pH suggests the coupling to a proton transfer process. Altogether, our data indicate a partially unfolded ferricytochrome c conformation with spectroscopic characteristics reminiscent of the recently discovered alkaline isomer V(b), which is stabilized under folding conditions by exposing the protein to a very alkaline pH for an extended period of time.