Modulation of Ligand-Field Parameters by Heme Ruffling in Cytochromes c Revealed by EPR Spectroscopy

Abstract

Electron paramagnetic resonance (EPR) spectra of variants of Hydrogenobacter thermophilus cytochrome c552 (Ht c-552) and Pseudomonas aeruginosa cytochrome c551 (Pa c-551) are analyzed to determine the effect of heme ruffling on ligand-field parameters. Mutations introduced at positions 13 and 22 in Ht c-552 were previously demonstrated to influence hydrogen bonding in the proximal heme pocket and to tune reduction potential (Em) over a range of 80 mV [Michel, L. V.; Ye, T.; Bowman, S. E. J.; Levin, B. D.; Hahn, M. A.; Russell, B. S.; Elliott, S. J.; Bren, K. L., Biochemistry 2007, 46, 11753–11760]. These mutations are shown here to also increase heme ruffling as Em decreases. The primary effect on electronic structure of increasing heme ruffling is found to be a decrease in the axial ligand-field term Δ/λ, which is proposed to arise from an increase in the energy of the dxy orbital. Mutations at position 7, previously demonstrated to influence heme ruffling in Pa c-551 and Ht c-552, are utilized to test this correlation between molecular and electronic structure. In conclusion, the structure of the proximal heme pocket of cytochromes c is shown to play a role in determining heme conformation and electronic structure.

Structural Characterization of Nitrosomonas europaea Cytochrome c‐552 Variants with Marked Differences in Electronic Structure

“…This analysis examined cyt c variants and demonstrated that those with the more ruffled hemes show greater rhombicity and lower g max values. [16] A similar relationship has been noted in cyanide derivatives of globins. [17] However, for a given system, the changes in g max brought about by changes in heme ruffling are relatively small, and might not account for the range of g max values observed.…”

“…[20] Analysis of EPR spectra of cyt c variants with different amounts of heme ruffling supports the model that increasing ruffling raises the d xy orbital energy relative to that of the dp orbitals, increasing rhombicity. [16] Cyts c with more ruffled hemes are thus predicted to have more rhombic EPR spectra, corresponding to lower g max values; the observation that cyanide complexes of some globins display HALS EPR signals for species with relatively planar hemes is consistent with this finding. [17] The determination of crystal structures of Ne c-552 variants here allows us to investigate the relationship between heme conformation and electronic structure further.…”

“…[17] However, for a given system, the changes in g max brought about by changes in heme ruffling are relatively small, and might not account for the range of g max values observed. [16] Recent studies utilizing Pseudomonas aeruginosa cyt c-551 (Pa c-551) and Nitrosomonas europaea cyt c-552 (Ne c-552) as model systems to investigate the structural basis for variations in EPR spectra of heme proteins with His/Met axial ligation showed that selected point mutations introduced in the heme pocket triggered large changes in the EPR spectra. [14,15] In particular, whereas the g max values for Pa c-551 and Ne c-552 are 3.20 and 3.34, respectively, the Pa N64V and Ne N64D variants have g max values of 3.05 and 3.13, respectively (Table 1, Figure 2).…”

Conformational control of cofactors in nature – the influence of protein-induced macrocycle distortion on the biological function of tetrapyrroles

“…In a follow-up focused upon delineating the influence of ruf distortion vs. Fe(III)-HIS bond strength on the EPR spectra of H. thermophiles cyt c 552 and Pseudomonas aeruginosa cyt c 551 , it was again suggested that heme ruffling was correlated with decreased reduction potentials. 260 However, here the results were confounded by the fact that the reduction potentials of the mutants could also be explained by a change in the Fe(III)-HIS bond strength. However, the effects were considered separable on the basis that the observed change in the EPR axial ligand field term was opposite to what would be expected from changes in the axial HIS bond strength.…”

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

“…The degree of ruffling will influence the electronic structure of the porphyrin [197,198] and has been shown to decrease delocalization of π electrons [199–205]. Inducing heme distortion by protein changes have shown that the E° can be changed by 170 mV in heme-nitric oxide/oxygen proteins [204] (Figure 16).…”