Fast Coordination Changes in Cytochrome c Do Not Necessarily Imply Folding

Abstract

Folding of globular proteins occurs with rates that range from microseconds to minutes; consequently, it has been necessary to develop new strategies to follow the faster processes that exceed stopped-flow capabilities. Rapid photochemical methods have been employed to study the rate of folding of reduced cytochrome c. In this protein, the iron of the covalently bound heme binds a His and a Met, proximal and distal. Unfolding by guanidine or urea weakens the Fe-Met bond, and the reduced unfolded cytochrome c easily binds CO and other heme ligands, which would react slowly or not at all with the native protein. Therefore in the presence of CO, reduced cytochrome c unfolds at lower denaturant concentrations than in the absence of this ligand, and rapid photochemical removal of CO from unfolded cytochrome c, is expected to trigger at least an incomplete refolding. This approach is complicated by the breakage of the proximal His-Fe bond that may occur as a consequence of CO photodissociation in the unfolded cytochrome c because of the so-called base elimination mechanism. Rebinding of CO to the four-coordinate heme yields kinetic intermediates unrelated to folding. Our hypothesis is supported by parallel observations carried out with protoheme and microperoxidase.

Effects of Heme on the Structure of the Denatured State and Folding Kinetics of Cytochrome b562

“…weakened by mutagenesis of the ligands. 36,37 It has been suggested that changes in co-ordination to the heme iron are not related to any folding events in cytochrome c. 38 In this current example of cytochrome b 562 , the changes in the protein fold and mobility that result in this weakening or loss of heme iron ligands appear to be limited mainly to the terminal stretches of sequence around these ligands, residues 1-15 and 88-106 (coloured yellow in state A in Figure 9). The folding of the ferrous holo protein 16,17 was initiated by reduction of the ferric protein in this compact state.…”

“…9 Notably, residues 3-15 and 88-100 (in the sequences around the ligand residues, 7 and 102 at either terminus of the protein) no longer gave daN(i,iC3) NOEs indicative of a-helical conformation. Additionally, there is a gap (residues [33][34][35][36][37][38] in the helical pattern corresponding to helix 2 of the native protein. Helix 3 (residues 62-82) of the native protein appeared completely intact in this species.…”

A heme tag for in vivo synthesis of artificial cytochromes

“…Furthermore, no peaks were detected at the position of the β or α band (523 nm or 550 nm, respectively). Heme ligands have previously been shown to influence the spectral behaviour in the 550-nm range (Arcovito et al 2001;Lee et al 2001). Upon addition of 300 mM imidazole to MP301, the dithionite-reduced spectrum resembled that of the other heme His 6 -tagged fusion proteins (Fig.…”

Fast Dissociation of Nitric Oxide from Ferrous Pseudomonas aeruginosa cd1 Nitrite Reductase

“…Different amounts (30 -90 M) of a NO solution were added anaerobically to produce the reduced NO-bound derivative. The instrument used for photolysis experiments has been described elsewhere (17) and uses a Nd-YAG solid-state laser with a 5-ns pulse (Quanta System HIL 101, second harmonic ϭ 532 nm, E ϭ 80 mJ/pulse) that was focused onto the filled cuvette containing the desired solution. The experimental data thus collected are time courses at single wavelength; they are converted to absorbances by means of the IGORPRO package (Wavemetrics).…”