Role of Ligand Substitution in Ferrocytochrome c Folding

Abstract

The ligand substitutions that occur during the folding of ferrocytochrome c [Fe(II)cyt c] have been monitored by transient absorption spectroscopy. The folding reaction was triggered by photoinduced electron transfer to unfolded Fe(III)cyt c in guanidine hydrochloride (GuHCl) solutions. Assignments of ligation states were made by reference to the spectra of the imidazole and methionine adducts of N-acetylated microperoxidase 8. At pH 7, the heme in unfolded Fe(II)cyt c is ligated by native His18 and HisX (X = 26, 33) residues. The native Met80 ligand displaces HisX only in the last stages of folding. The ferroheme is predominantly five-coordinate in acidic solution; it remains five-coordinate until the native methionine binds the heme to give the folded protein (the rate of the methionine binding step is 16 +/- 5 s-1 at pH 5, 3.2 M GuHCl). The evidence suggests that the substitution of histidine by methionine is strongly coupled to backbone folding.

Rate of intrachain contact formation in an unfolded protein: temperature and denaturant effects

“…We obtained the methionine-heme spectrum by adding B 1 (l) to the known deoxyheme spectrum until the result generated the best ®t to reference Fe(II) heme-methionine spectra; we obtained reference spectra from our measurements of native cytochrome c and from published sources. 3,4,42 We found very good agreement between the observed transient spectra and the ®t to Scheme I. The slow t 4 process, which is presumably related to refolding/unfolding kinetics omitted from Scheme I, erodes the quality of ®ts that include millisecond time-scales (although it does not affect our analysis of the very fast heme-Met80 loop kinetics).…”

“…Results agreed with published Fe(II) hemehistidine spectra. 3,4,42 The methionine-liganded heme spectrum is also a ®xed input, obtained as follows: The fastest exponential decay t 1 in the data corresponds to the replacement of the deoxyheme by the methionine complex. Thus B 1 (l) is proportional to the deoxyheme minus methionine-heme difference spectrum.…”

Cytochrome c ′ folding triggered by electron transfer: Fast and slow formation of four-helix bundles

“…The absence of strongly shifted peaks in the 1 Fe II -Cyt c Folding. Fe II -Cyt cЈ folding was initiated by rapid electron injection (Ϸ100 s) into unfolded oxidized protein ([GuHCl] ϭ 2.02-2.54 M) after two-photon laser excitation of NADH (43,44). Under these conditions, heme reduction is slower than binding of the nonnative sixth ligand.…”

Heme Ligation and Conformational Plasticity in the Isolatedc Domain of Cytochrome cd 1 Nitrite Reductase

“…Electrochemical examination has revealed that the oxidized His 18 -His 82 form is highly disordered, and it is proposed that this high level of disorder facilitates rapid rearrangement to His 18 -Met 80 upon reduction (8). Heme-ligand exchange reactions between methionine and histidine residues were first observed during the in vitro folding and unfolding reactions of oxidized (9 -11) and reduced (12,13) horse heart cytochrome c. His 18 and Met 80 are the axial heme ligands in native cytochrome c in both oxidation states. In unfolded cytochrome c, the proximal histidine (His 18 ) remains an axial ligand by virtue of its proximity to Cys 15 and Cys 17 , which form the thioether linkages to the porphyrin ring (14 -16).…”