PROTEIN-INDUCED CHANGES IN NONPLANARITY OF THE PORPHYRIN IN NICKEL CYTOCHROME-C PROBED BY RESONANCE RAMAN-SPECTROSCOPY

The influence of the protein on the nonplanarity of the macro cycle fo r nickel(II)-reconstituted cytochrome c (NiCyt-c) has been investigate d with pH-dependent resonance Raman and UV-visible absorption spectros copy and molecular mechanics calculations. The spectra reveal that NiC yt-c near neutral pH has axially coordinated Ni, but below pH 3 and ab ove pH 12, four-coordinate species predominate. The shape of the struc ture-sensitive Raman line upsilon(10) of NiCyt-c is asymmetric and bro ad and it changes with pH. This broad line can be decomposed well into at least two sublines, a low-frequency line that results from a nonpl anar conformer and a high-frequency line that arises from a nearly pla nar conformer. relative to that of the planar conformer. Upon lowering the pH from 3.0 to 1.0, the amount of the nonplanar conformer decreas es relative to that of the planar conformer. The decreased nonplanarit y can be accounted for in terms of the disruption of a hydrogen-bondin g network in the peptide backbone upon lowering the pH. Molecular mech anics (MM) calculations on iron(III) and nickel(II) microperoxidase 5 (MP-5) as well as some model heme derivatives have been carried out in order to locate the part of the protein that causes the heme distorti on observed in the X-ray crystal structures of cytochromes c. The ener gy-optimized structures of MP-5 and the model compounds were analyzed using the normal-coordinate structural decomposition method to specify and quantify the out-of-plane macrocyclic distortions. MM calculation s for MP-5 show that two hydrogen bonds formed between the amide group s in the peptide backbone are important in maintaining the ruffled def ormation of the macrocycle. All evidence presented supports the hypoth esis that the nonplanar distortion of the porphyrin of cytochromes c i s largely maintained by a relatively small protein segment including t he cysteines, the amino acids between the cysteines, and the adjacent histidine ligand. Hydrogen bonding within the backbone of this segment is important in maintaining the conformation of the peptide that indu ces the porphyrin distortion.