Variation and Analysis of Second-Sphere Interactions and Axial Histidinate Character inc-type Cytochromes

Abstract

The electron-donating properties of the axial His ligand to heme iron in cytochromes c (cyts c) are found to be correlated with midpoint reduction potential (E m ) in variants of Hydrogenobacter thermophilus cytochrome c 552 (Ht cyt c 552 ) in which mutations have been made in and near the Cys-X-X-Cys-His (CXXCH) heme-binding motif. To probe the strength of the His-Fe(III) interaction, we have measured 13 C nuclear magnetic resonance (NMR) chemical shifts for 13 CN − bound to heme iron trans to the axial His in Ht Fe(III) cyt c 552 variants. We observe a linear relationship between these 13 C chemical shifts and E m , indicating that the His-Fe(III) bond strength correlates with E m . To probe a conserved hydrogen bonding interaction between the axial His Hδ1 and the backbone carbonyl of a Pro residue, we measured the pK a of the axial His Hδ1 proton (pK a(2) ), which we propose to relate to the His-Fe(III) interaction, reduction potential and local electrostatic effects. The observed linear relationship between the axial His 13 Cβ chemical shift and E m is proposed to reflect histidinate (anionic) character of the ligand. A linear relationship also is seen between the average heme methyl 1 H chemical shift and E m which may reflect variation in axial His electron-donating properties or in the ruffling distortion of the heme plane. In summary, chemical shifts of the axial His and exogenous CN − bound trans to His are shown to be sensitive probes of the His-Fe(III) interaction in variants of Ht cyt c 552 and display trends that correlate with E m .

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

“…19 For a given His orientation, histidinate character also is related to the strength of the hydrogen bond between the axial His δ1 NH and its hydrogen bond acceptor, which in cytochromes c is a proline carbonyl. Because a stronger bond from His to Fe(III) stabilizes the higher oxidation state, reduction potential will decrease with increasing His-Fe(III) bond strength.…”

“…Mutation of residue 7 has been shown to influence heme ruffling in both proteins, 11, 16, 17 and mutations of residues 13 and 22 have been shown to affect the His-Fe(III) interaction and are proposed to influence heme ruffling. 18, 19 …”

Chemical reactivity of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

“…The two thioether bonds in c -type cytochromes are formed from a CXXCH motif, the role of which has been discussed extensively [73, 76, 77]. An XXXCH signature, leading to the formation of a single thioether bond, is recognizable in mitochondrial cytochromes c and c 1 from trypanosomatids and euglenids [78].…”

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

“…These mutations are discussed in more details in section 5.4.2. Interestingly, strengthening the H-bond to the proximal His ligand of cytochrome c resulted in a 100 mV decrease in redox potential [240]. Disruption of H-bonds from Tyr to the Met axial ligand in yeast iso-1 cytochrome c resulted in a decrease of up to 56 mV in E°.…”