Fe- but not Mg-protophorphyrin IX binds to a transmembrane b-type cytochrome

Figures & data

Figure 1. Purification and in vitro reconstitution of holo-cytochrome b₆. (A) SDS-PAGE analysis (10% SDS-gel) of the apo-cytochrome b₆ expression and purification. 1: Cell extract before induction; 2: total cellular extract; 3: soluble proteins; 4: inclusion bodies; 5: affinity purified apo-cytochrome b₆ fused to MBP. (B, C) Absorbance spectra show specific binding of heme to apo-cytochrome b₆ under oxidizing (B) and reducing (C) conditions. Vis spectra of free heme (dark grey) and 150 μg in vitro reconstituted apo-cytochrome b₆ (grey) are shown. The inset in Figure (C) shows the redox difference spectrum.

Figure 2. Chlorophyll a does not associate with apo-cytochrome b₆. Absorbance spectra of free chlorophyll a (dark grey) and chlorophyll a after addition of 150 μg cytochrome b₆ (grey) recorded under oxidizing conditions.

Table 1. Absorbance maxima offree protoporphyrin IX derivatives (without cyt b₆) and in presence ofcytochrome b₆ (with cyt b₆) are listed. The bandwidth at half maximum (fwhm) is given for the Soret band (γ-band) of each spectrum. Absorbance maxima and the fwhm values were obtained after Gaussian deconvolution of the respective absorbance curve using the program PeakFit (Systat Software, Inc.).

		Without cyt b6		With cyt b6
Porphyrin	Redoxstate	Maximum	Fwhm	Maximum	Fwhm
Fe-protoporphyrinI X (heme)	Oxidized	405	32.7	414	26
		606		531
				562
	Reduced	418	39.8	429	22
		541		532
		569		562
Chlorophyll a	Oxidized	378		378
		416		416
		439	24.7	439	23.4
		588		590
		626		627
		671	25.7	671	24.5
Protoporphyrin IX	Oxidized	408	38.2	408	39
		506		506
		541		541
		578		578
		632		632
Mg-protoporphyrin IX	Oxidized	419	23.7	419	23.3
		551		551
		588		588
Fe-deuteroporphyrin IX	Oxidized	401	33.5	409	33
		569		570
		608		605
	Reduced	408	34.3	411	33.8
		520		525
		554		549
Fe-protoporphyrin IX dimethylester	Oxidized	395	22.5	400	23
		488		524
		592
	Reduced	409	39.9	425	21.7
		569		530
		606		559

Figure 3. Heme but not chlorophyll co-elutes with cytochrome b₆ after in vitro reconstitution. Free heme and chlorophyll a, respectively, were added to isolated apo-cytochrome b₆ and the protein was purified via Ni-NTA chromatography. After elution of the protein, absorbance spectra were recorded under oxidizing (dark grey) and reducing conditions (grey) in case of heme. Chlorophyll a did not elute from the column together with the protein, therefore a chlorophyll a spectrum is not visible (light grey).

Figure 4. Apo-cytochrome b₆ does not bind porphyrins with central and porphyrins in presence of 145 μg apo-cytochrome b₆ (grey) and porphyrins in presence of 145 mg apo-cytochrome b₆ (grey) are show nunder oxidizing conditions. (A) ProtoporphyrinIX, (B) Mg-protoporphyrin IX.

Figure 5. Chemical structures ofheme, chlorophyll a and protoporphyrin IX derivatives used in this study. Arrows indicate side chains varied compared to heme. In Fe-deuteroporphyrin IX ethylene side chains 2 and 4 are substituted for hydrogen atoms whereas in Fe-protoporphyrin IX dimethylester the propionyl groups 6 and 7 are esterified with methyl groups.

Figure 6. Apo-cytochrome b₆ binds porphyrins with modified side chains. Absorbance spectra of Fe-deuteroporphyrin IX (A, B) as well as of Fe-protoporphyrin IX dimethylester (C, D) were recorded under oxidizing (A, C) and reducing (B, D) conditions. The spectrum of free porphyrin (dark grey) is compared to the spectrum of the porphyrin after addition of 150 μg apo-cytochrome b₆ (grey). The insets in (B) and (D) are redox difference spectra.

Figure 7. Porphyrin binding isotherms for cytochrome b₆. Increasing amounts ofapo-cytochrome b₆ were titrated into buffer containing either 5 μM heme (squares), Fe-deuteroporphyrin IX (circles) or Fe-protoporphyrin IX dimethylester (triangles). The protein-to-heme ratio was plotted against the normalized absorbance change at 413 nm. Each data point represents an individual measurement.