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. 28:768-773.

2. Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76:2879-2886.

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.

(A)

(C)

(B)

ΔTGEE rHO-1

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 450-500 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.

(A)

(B)

(D)

(C)

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.

(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.

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.

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.

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.

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.

m

free

Table S1 Data collection and refinement statistics

ΔTGEE–heme–rHO-1 complex

Data collection

Space group P61

Cell dimensions (Å) a = b = 290.3, c = 83.6

Resolution (Å) 50 – 4.3 (4.37-4.30) *

Rsy a 0.107 (0.888)

I / σI 6.2 (2.8) Completeness (%) 99.9(100) Redundancy 12.0 (10.3)

Refinement

Resolution (Å) 41.1– 4.3

No. reflections 26421 Rwork / R b 0.22 / 0.26 No. atoms

Protein 13038

Ligand 316

B-factors

Protein 235.0 Ligand 234.8

R.m.s. deviations

Bond lengths (Å) 0.012

Bond angles (°) 1.801

*Values in parentheses are for highest-resolution shell.

aRsym = ΣhklΣi |Ii(hkl) - <I(hkl)>| / ΣhklΣiIi(hkl), <I(hkl)> is the mean

intensity for multiple recorded reflections. bRwork = Σ|Fobs(hkl) - Fcalc(hkl)| / Σ|Fobs(hkl)|. Rfree is the Rcryst calculated for

the five percent of the dataset not included in the refinement.

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.