The Influence of Heme Ruffling on Spin Densities in Ferricytochromes c Probed by Heme Core ¹³C NMR

Abstract

The heme in cytochromes c undergoes a conserved out-of-plane distortion known as ruffling. For cytochromes c from the bacteria Hydrogenobacter thermophilus and Pseudomonas aeruginosa, NMR and EPR spectra have been shown to be sensitive to the extent of heme ruffling and to provide insights into the effect of ruffling on electronic structure. Using mutants of each of these cytochromes that differ in the amount of heme ruffling, NMR characterization of the low-spin (S=1/2) ferric proteins has confirmed and refined the developing understanding of how ruffling influences spin distribution on heme. The chemical shifts of the core heme carbons were obtained through site-specific labeling of the heme via biosynthetic incorporation of 13C-labeled 5-aminolevulinic acid derivatives. Analysis of the contact shifts of these core heme carbons allowed Fermi contact spin densities to be estimated, and changes upon ruffling to be evaluated. The results allow a deconvolution of contributions to heme hyperfine shifts and a test of the influence of heme ruffling on electronic structure and hyperfine shifts. The data indicate that as heme ruffling increases, the spin densities on the β-pyrrole carbons decrease, while the spin densities on the α-pyrrole carbons and meso carbons increase. Furthermore, increased ruffling is associated with stronger bonding to the heme axial His ligand.

Fixation of Zinc(II) Ion to Dioxygen in a Highly Deformed Porphyrin: Implications for the Oxygen Carrier Mechanism of Distorted Heme

“…13 This distortion is energetically unfavorable, so it universality suggests that such conformational variation is crucial to the functions of metalloporphyrins and heme. 7,14 Nonplanar metalloporphyrins show many interesting properties, including potent catalytic ability, 15 variable coordination, 16 large spectral red shift, 17 tunable spin densities, 18 and other unusual physical properties. 19 In heme chemistry, the electronic structure of the metal ion in a complex can undergo marked changes induced by a slight variation of the macrocycle.…”

Going with the Electron Flow: Heme Electronic Structure and Electron Transfer in Cytochrome c

“…Thus, increasing heme planarity is predicted to enhance electronic coupling with redox partners that dock near the exposed heme pyrrole. A follow‐up study by Kleingardner examining the chemical shifts of heme core 13 C nuclei verified these conclusions and also provided new insights into the effects of ruffling on the pseudocontact shift and on spin polarization …”

“…We also identified mutations in the distal pocket that impact ruffling, although some of these mutations alsoh ave an impact on the distal Metl igand orientation (Figures5Aa nd 5B). Changes in ruffling were assessed using X-ray crystallography [67,68] and/or through analysis of hyperfine shiftsi nN MR spectra [67,93,101,102] (Figure 7) and g values measured by EPR. [67,[103][104][105] In experiments,w eobserved ac orrelation between enhanced ruffling and lowered reduction potential when comparing wild-type and mutant proteins.…”

“…Ther esults reveal that the A7F mutation decreases heme ruffling by up to~0.1 , and this decrease in ruffling is accompanied by an in-crease of distribution of unpairede lectrons pin density to the porphyrin periphery.T hus,i ncreasing hemep lanarity is predicted to enhance electronic coupling with redox partners that dock near the exposed heme pyrrole.A follow-up studyb yK leingardner examiningt he chemical shifts of heme core 13 Cn uclei verified these conclusions and also provided new insights into the effects of ruffling on the pseudocontact shift and on spin polarization. [101] To test the hypothesis that increasing the delocalization of the redox-active molecular orbital to the heme edge enhances ETr ates,C hampion examined photoreduction cross-sections,c orresponding to ET rates,b yt ransient absorption spectroscopy on Pa c-551, Pa-F7A, and horse heart cyt c. [124] Previousw ork showed that nearbya romatic amino acid residues are electron donors in heme photoreduction, [125] and through analysis of the heme pocket structures of Pa c-551 and Pa-F7A, the likely electron donor in both proteins was determined to be Ty r27, which lies 3.5 from the hemee dge.I nh orse cyt c,t he likely electron donor is Ty r67, which is 3.3 from the heme edge.T hese threep roteins show differences in photoreduction cross-sections (corresponding to ET rates) spanningo ver two orders of magnitude,d espite their similar reduction potentials and D-A distances ( Figure 10A). Them ost efficient photoreductionw as observed for Pa c-551, followed by Pa-F7A and horse cyt c,i.e., in the order of increased ruffling.…”

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c 552 and its b-type variant

“…These include changes for Lys 6 and Lys 8 in helix 1, Gly 22 and Tyr 25 in the loop between helices 1 and 2, His 14 and Met 59 which form the axial ligands to the heme group and Val 53, which forms hydrogen bonds to a heme propionate group in the X-ray structure WT HT- c 552 . All these residues are in the vicinity of the heme-binding pocket and the differences may reflect changes in out-of-plane deformations of the heme group (Kleingardner et al 2013 ; Sun et al 2014 ). The changes for the axial ligand His 14 could also result from changes in the His-Fe interaction, a factor which has been suggested to play a role in tuning heme redox potentials (Bowman and Bren 2008 ; Michel et al 2007 ), or changes in the CXXCH loop stiffness (Galinato et al 2015 ).…”