<i>In vitro</i> reconstitution of cyt. b₆ and its G_n-mutants containing prolonged BC loops.

<p>Absorbance spectra of <i>in vitro</i> reconstituted cyt. b₆ (black), cyt. b₆-G₅ (dark gray) and cyt. b₆-G₁₀ (light gray) under oxidizing (A) and reducing (B) conditions. G₅ and G₁₀ stands for the number of Gly residues inserted into the BC-loop. The absorbance maxima are essentially identical for all three proteins (A: 561 nm / 532 nm / 414 m; B: 562 nm / 532 nm / 429 nm for the α-/β-/γ-band). The same applies for the redox difference spectra (562 nm / 532 nm for the α-/β-band) (inlet B). (C) and (D) show titrations of cyt. b₆ (black squares), cyt. b₆-G₅ (dark gray spheres) and cyt. b₆-G₁₀ (light gray triangles) with heme (C) or SDS (D) under oxidizing conditions. Each data point represents an individual measurement. The quotient of the absorbance intensity at 414 nm to 404 nm was calculated, representing the absorbance maximum of the Soret-band of bound and unbound heme, respectively, and normalized to “0” for solely free heme and “1” for solely bound heme. (C) Increasing amounts of protein were titrated into buffer containing 5 μM heme to measure heme-binding isotherms. For all three proteins, the titration experiments show a saturation at a protein/heme ratio of 0.5 and higher. (D) To determine the proteins´ stability, increasing amounts of SDS were added to reconstituted protein. All three cyt. b₆ variants are stable up to an SDS concentration of ~4.5 mM.</p

Purification of cf-cyt. b₆ and the two cyt. b₆ halves cf-cyt. b₆-AB and cf-cyt. b₆-CD, and assembly of the cyt. b₆ holo-protein.

<p>(A) Proteins were separated on an 18% SDS gel. M: molecular mass standard; P: cell-free expressed protein; AC: protein purified by affinity chromatography. (B to E) UV/VIS absorbance spectra were acquired under oxidizing (black) and reducing (gray) conditions. The inlet shows the redox difference spectrum. (B) Cf-apo-cyt. b₆ (3 μM) was mixed with heme (6 μM. (C) cf-cyt. b₆-AB was mixed with cf-cyt. b₆-CD in a one-to-one ratio (1 μM each), and heme (2 μM) was added in total protein to a heme ratio of one to one. (D) cf-cyt. b₆-AB (2 μM) was mixed with heme (2 μM) and (E) cf-cyt. b₆-CD (2 μM) was mixed with heme (2 μM). Note that different concentrations of protein and heme were used in (B) compared to (C), (D) and (E). The spectra in (B), (C) and (D) show the typical cyt. b₆ absorbance maxima (ox: 562 nm / 531nm / 414 nm, red: 562 nm / 532 nm / 429 nm, redox: 562 nm / 532 nm for the α-/β-/γ-band).</p

Absorbance spectra of cyt. b₆ after proteolysis.

<p>(A, B) Holo-cyt. b₆ was proteolytically digested with proteinase K. At defined time points (10 min light blue, 30 min green, 1 h yellow, 2 h red, 5 h purple) proteolysis was stopped and absorbance spectra were measured under oxidizing (A) and reducing conditions (B). As a control, the absorbance spectra of free heme (black) and reconstituted, undigested cyt. b₆ (dark blue) were measured. The color legend in (A) also applies to (B). All spectra show the cyt. b₆ characteristic maxima (ox: 562 nm / 531nm / 414 nm, red: 562 nm / 532 nm / 429 nm, for the α-/β-/γ-band).</p