Electron Paramagnetic Resonance Characterization of the Triheme Cytochrome from <i>Geobacter sulfurreducens</i>

Periplasmic cytochrome A (PpcA) is a representative of a broad class of multiheme cytochromes functioning as protein “nanowires” for storage and extracellular transfer of multiple electrons in the δ-proteobacterium <i>Geobacter sulfurreducens</i>. PpcA contains three bis-His coordinated hemes held in a spatial arrangement that is highly conserved among the multiheme cytochromes c₃ and c₇ families, carries low potential hemes, and is notable for having one of the lowest number of amino acids utilized to maintain a characteristic protein fold and site-specific heme function. Low temperature X-band electron paramagnetic resonance (EPR) spectroscopy has been used to characterize the electronic configuration of the Fe­(III) and the ligation mode for each heme. The three sets of EPR signals are assigned to individual hemes in the three-dimensional crystal structure. The relative energy levels of the Fe­(III) 3d orbitals for individual hemes were estimated from the principal <i>g</i>-values. The observed <i>g</i>-tensor anisotropy was used as a probe of electronic structure of each heme, and differences were determined by specifics of axial ligation. To ensure unambiguous assignment of highly anisotropic low-spin (HALS) signal to individual hemes, EPR analyses of iron atom electronic configurations have been supplemented with investigation of porphyrin macrocycles by one-dimensional ¹H NMR chemical shift patterns for the methyl substituents. Within optimized geometry of hemes in PpcA, the magnetic interactions between hemes were found to be minimal, similar to the c₃ family of tetraheme cytochromes