Protonation of the Cysteine Axial Ligand Investigated in His/Cys c-Type Cytochrome by UV–Vis and Mid- and Far-IR Spectroscopy

Abstract

His/Cys coordination was recently found in several c-type cytochromes, which could act as sensors, in electron transport or in regulation. Towards better understanding Cys function and reactivity in these cytochromes, we compare cytochrome c6 (c6wt) from the cyanobacterium Nostoc PCC 7120 with its Met58Cys mutant. We probe the axial ligands and heme properties by combining visible and Mid to Far FTIR difference spectroscopies. Cys58 determines the strong negative redox potential and pH dependence of M58C (EmM58C =-375 mV, versus Emc6wt = +339 mV). Mid-IR (notably Cys (SH), His (C5N1), heme (CmH)) and Far-IR ((Fe(II)-His), (His-Fe(III)-Cys)) markers of the heme and ligands show that Cys58 remains a strong thiolate ligand of reduced Met58Cys at alkaline pH, while it is protonated at pH 7.5, stabilized by a strong hydrogen bonding interaction, and weakly interacts with Fe(II). These data provide a benchmark for further analysis of c-type cytochromes with natural His/Cys coordination.

A repurposed, non-canonical cytochrome c, chaperones calcium binding by PilY1 for type IVa pili formation

“…The presence of cysteine-to-Fe 3+ charge transfer bands at ~630 nm and ~730 nm in the oxidised form, which disappear upon dithionite reduction (Fig. 5b, inset), are also in full agreement with spectral properties of His/Cys coordinated c-type cytochromes 35 . To further support that the special spectral properties are due to Cys 91 , we purified the MalE-TfcP C91M variant (Fig.…”

“…This fits well to spectra of canonical cytochromes c. Interestingly, we did not observe a red-shift of the Soret-peak from the oxidised to the reduced spectrum. For canonical cytochromes c with His/His or His/Met coordination a 10 nm bathochromic shift is typically observed, while a semisynthetic cytochrome c with His/Cys coordination of the heme iron did not exhibit this shift 35,41,42 , suggesting that Cys (Fig. 1cd) is the second axial ligand in TfcP and responsible for the lack of the red-shift.…”

“…Interestingly, in Mxan_0363 and homologs, this is a Cys residue (Cys 91 in Mxan_0363) (Fig. 1cd), which is rarely found as the second axial ligand in c type cytochromes 34,35 . In the vicinity of Cys 91 , no conserved Met or His residues are present.…”

“…5d) that fit well to the gvalues observed for multiple heme-containing proteins with a cysteine thiolate ligated heme iron 44 . Cytochromes c with a distal Cys were reported to have a very low midpoint potential in the range of -350 mV while canonical cytochromes c have a potential of approximately +250 mV 35,41,42,45 . To determine whether TfcP has a similarly low redox-potential, we used UV-Vis and EPR monitored redox titrations.…”

“…the triheme DsrJ of Allochromatium vinosum, are involved in dissimilatory sulfur metabolism in which sulfate is used as terminal electron acceptor 56 . Other c-type cytochromes with His/Cys ligation have been suggested to have a role in signalling 35,57 .…”