Modulation of the Ligand-Field Anisotropy in a Series of Ferric Low-Spin Cytochrome c Mutants derived from Pseudomonas aeruginosa Cytochrome c-551 and Nitrosomonas europaea Cytochrome c-552: A Nuclear Magnetic Resonance and Electron Paramagnetic Resonance Study

Abstract

C-type cytochromes with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and over-expressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand that have a considerable impact on both heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by lowtemperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. The Ne c-552 has a ferric low spin (S=1/2) EPR signal characterized by large g anisotropy with g max resonance at 3.34; a similar large g max value EPR signal is found in the mitochondrial Complex III cytochrome c 1 . In Ne c-552, deletion of Asn64 (NeN64Δ) changes the heme ligand-field from more axial to rhombic (small g anisotropy and g max at 3.13) and furthermore hinders the Met fluxionality present in the wild-type enzyme. In Pa c-551 (g max at 3.20) replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g max at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Δ) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Δ, NeG50N/V65Δ, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak axial (Pa c-551) field, but had an impact on Met orientation, fluxionality and relaxation dynamics. Comparison of the electronic fingerprints in the over-expressed proteins and their mutants reveals a linear relation between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III) the large g max value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.

Cytochrome c: A Multifunctional Protein Combining Conformational Rigidity with Flexibility

“…A large rhombic distortion in heme proteins with this spin and ligation state is likely to result from static Jahn-Teller distortions which are facilitated by theground state of the heme iron. The existence of a rather strong rhombic deformation in ferric cytochrome c species is confirmed by EPR data, which allowed for the discovery of two types of ferric cytochrome c proteins: type I with a negligible and type II with a very strong rhombic splitting of the , energies [30]. Apparently, horse heart cytochrome c belongs to the type II category.…”

“…The destabilization of the -orbital ( 2 ) is a result of the capability of 1 -type ruffling to mix with the 2 ( ) orbital. Based on EPR and NMR investigations of Pseudomonas aeruginosa cytochrome c 551 and Nitrosomonas europaea cytochrome c 552 , Zoppelaro et al proposed a correlation between rhombic ( 1 ) deformations and ruffling ( 1 ) [30]. As shown in a more recent paper from our group, each type of out-of-plane deformation can indeed induce in-plane deformation of the porphyrin macrocycle described by [72] …”

“…In spite of a rather asymmetric heme environment and strong heme-protein interactions via the two axial ligands [10,30,31], the thioether bridges [32], the internal electric field at the heme [33][34][35][36][37][38], and to a lesser extent substituent-hydrogen bonding [25,26,[39][40][41] and multiple van der Waals contacts [42], spectroscopists have for a long period of time considered the heme as exhibiting an ideal D 4h -symmetry. This was (and in part still is) particularly true for the interpretation of optical absorption and resonance Raman data [43][44][45][46][47][48][49].…”

“…This was (and in part still is) particularly true for the interpretation of optical absorption and resonance Raman data [43][44][45][46][47][48][49]. This view ignores very early EPR data for many paramagnetic heme proteins including ferricytochrome c that revealed a rhombic deformation of the heme iron's ligand field, which reflects deformations of the heme group assignable to the irreducible representation 1 of the D 4h -point group [10,30,50]. Such a deformation lowers the overall symmetry of the heme to C 2v .…”

“…Such a deformation lowers the overall symmetry of the heme to C 2v . Chemical shift distributions in, for example, 1 H NMR spectra, of methyl groups revealed the nonequivalence of the methyl substituents of the heme and thus a heme symmetry much lower than D 4h [30,51,52]. Senn et al reported NMR-data that revealed a rather asymmetric spin density distribution of the heme macrocycle in a cytochrome c derivative [53].…”