SI Text S1 Solution Scattering Data Collection and Analysis. SI references

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1 SI Text S1 Solution Scattering Data Collection and Analysis. The X-ray photon energy was set to 8 kev. The PILATUS hybrid pixel array detector (RIGAKU) was positioned at a distance of 606 mm from the sample. ΔTGEE heme rho-1 purified with the size-exclusion chromatography as described in Methods was concentrated up to 10 mg/ml. Scattering profile simulations from the crystal structure were carried out using CRYSOL (1). Ab initio dummy model was constructed with DAMMIN (2). SI references 1. Svergun D, Barberato C, Koch MHJ (1995) CRYSOL - a Program to Evaluate X- ray Solution Scattering of Biological Macromolecules from Atomic Coordinates. J. Appl. Crystallogr. 28: Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76:

2 Fig. S1 Confirmation of the generation of biliverdin-iron chelate during the single turnover reaction of heme-rho-1 supported with NADPH ΔTGEE system. The spectrum was recorded 70 min after the addition of desferrioxamine (final conc. 2 mm) into the reaction mixture at 220 min after the addition of NADPH at 25 C (Fig. 1B). Characteristic peak of biliverdin around 670 nm was observed.

3 (A) (B) ΔTGEE rho-1 (C) Fig. S2 Characterization of the co-eluted fraction shown in Fig. 3. (A) Absorption spectrum and (B) SDS/PAGE stained with Coomassie Brilliant Blue of the fraction (47.5 ml fraction in Fig. 3) co-eluted from the size-exclusion column onto which a mixture of ΔTGEE and heme rho-1 was applied. The absorption peak at 405 nm and the shoulder around nm represent heme rho-1 complex and ΔTGEE, respectively. (C) Elution profile of a mixture of ΔTGEE and apo rho-1 from size exclusion column. First and second peaks were eluted at 50.9 ml and 58.6 ml, respectively. The experimental conditions were the same as described in the legend for Fig. 3.

4 (A) (B) (C) (D) Fig. S3 Electron density of ΔTGEE heme rho-1 complex. (A) Cα trace of two independent ΔTGEE heme rho-1 complexes was superimposed on the electron density contoured by 1.0 σ. FMN and FAD domains, and rho-1 were colored yellow, orange, and magenta, respectively. Electron density and Cα trace were shown as a stereo diagram. (B) Close-up view of heme and FMN. Omit map of heme, FMN, FAD, and NADP + contoured by 3.0 σ (green) was also superimposed on the electron density (gray) and Cα trace. (C) Close-up view of FAD and NADP +. (D) Difference anomalous Fourier map calculated with the data obtained using 1.5 Å wavelength X-ray. Difference anomalous map (white) contoured by 4.5 σ was superimposed onto the ribbon model of the complex. Magenta and yellow chains showed ΔTGEE, whereas green and blue chains showed heme rho-1. Anomalous peaks were observed at the heme irons.

5 (A) (B). Fig. S4 Comparison of the crystal structure and SAXS result. (A) Experimental X-ray scattering curve from ΔTGEE heme rho-1 complex (solid line) and theoretical curve estimated from the crystal structure (dotted line). Radius of gyration from Guinier approximation was 3.05 nm, which is similar to that obtained from the crystal structure (2.92 nm). (B) Superimposition of the ribbon model of ΔTGEE heme rho-1 complex onto the ab initio dummy atom model obtained from SAXS result.

6 Fig. S5 Superimposition of ΔTGEE heme rho-1 onto ΔTGEE (PDB ID: 3ES9). ΔTGEE and rho-1 in ΔTGEE heme rho-1 was colored as yellow and pink, respectively. The least extended form of ΔTGEE (Mol A) was colored green. Other extended forms, Mols B and C, were shown in cyan and magenta, respectively. Only the co-factors of ΔTGEE in ΔTGEE heme rho-1 complex were shown for clarity. All FAD domains were fitting well, whereas FMN domain in each ΔTGEE showed different arrangements.

7 Fig. S6 Introduction of Cys mutations for formation of intermolecular disulfide bonds between CPR and rho-1. FMN and FAD domains and rho-1 were colored with yellow, orange, and pink, respectively. Mutated sites were shown as cyan stick models. FMN, FAD and heme were shown as blue stick models.

8 Fig. S7 Western blot analysis of artificial disulfide bond formation between CPR and heme rho-1 with anti-cpr or anti-rho-1 antibodies. SDS/PAGE was performed without 2-mercaptoethanol.

9 Fig. S8 Superimposition of ΔTGEE heme rho-1 onto the FMN and heme domains of cytochrome P450 BM3 (PDB ID: 1BVY). Superimposition was done so as to minimize the root-mean-square difference of FMN molecule. ΔTGEE heme rho-1 was colored as in Fig. 4. Ribbon model of FMN and heme domains of cytochrome P450 BM3 were yellow-green and red, respectively. FMN and heme in cytochrome P450 BM3 were shown as cyan stick models.

10 Table S1 Data collection and refinement statistics ΔTGEE heme rho-1 complex Data collection Space group P6 1 Cell dimensions (Å) a = b = 290.3, c = 83.6 Resolution (Å) ( ) * a R sy m (0.888) I / σi 6.2 (2.8) Completeness (%) 99.9(100) Redundancy 12.0 (10.3) Refinement Resolution (Å) No. reflections R work / R b free 0.22 / 0.26 No. atoms Protein Ligand 316 B-factors Protein Ligand R.m.s. deviations Bond lengths (Å) Bond angles ( ) *Values in parentheses are for highest-resolution shell. a R sym = Σ hkl Σ i I i (hkl) - <I(hkl)> / Σ hkl Σ i I i (hkl), <I(hkl)> is the mean intensity for multiple recorded reflections. b R work = Σ F obs (hkl) - F calc (hkl) / Σ F obs (hkl). R free is the R cryst calculated for the five percent of the dataset not included in the refinement.

11 Table S2. Oligonucleotide sequences to produce ΔTGEE and Cys-introduced mutants of CPR and rho-1. ΔTGEE-f GGCTTCTACCCCAAAGAACTC ΔTGEE-r TCGAGCATTCGCCAGTATGAG T88C-CPR-f CAGTGTGGAACCGCTGAGGAG T88C-CPR-r GGAGCCATAGAATACGATAATG Q517C-CPR-f TCTTGTTTCCGCTTGCCTTTCAAG Q517C-CPR-r TTTGCGCACGAACATGGGTAC V146C-HO-f CAGTGCCTGAAGAAGATTGCGC V146C-HO-r ACCCCCTGAGAGGTCACC K177C-HO-f ACCTGTTTCAAACAGCTCTATCGTG K177C-HO-r GGGGTTGTCGATGCTCGG The sites for Cys-introduced mutations were underlined.