P HOTOIRRADIATION ASSAY

The system of photoirradiation was constructed by using reconstituted metallo-porphyrin (ZnPP or ZnPE1)-Mbs as photo-sensitized enzymes (ZnPP-Mb or ZnPE1-Mb), the triethanolamine (TEA) as an electron donor, and nicotinamide adenine dinucleotide phosphate (NADP⁺) as an electron acceptor. This is a simplemodel of photosystem I that we can study the photo-induced NADP⁺ conversion efficiency and reaction rate within 6 hours photoirradiation of different metallo-porphyrins and mutated myoglobins. This experiments were carried out with 10 μM ZnPP-Mb (or ZnPE1-Mb), 2.5 mM NADP⁺, and 1 M TEA in 100 mM KPi pH 9.0 under room temperature. Before photoirradiation, there was no absorbance around 340 nm. Upon photoirradiation (λex = 419 nm) for 1 hour, the absorbance at 340 nm increased which was attributed to the reduction of NADP⁺ to form NADPH. Fig. 4-14

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and 4-15 revealed that the UV-Vis spectrum changed of ZnPP-Mb (or ZnPE1-Mb) was photoirradiated for 6 hours then kept in the dark for 5 days under room temperature.

We also used dark reaction (the same condition with photoirradiation but without irradiation of light) of ZnPP-Mb (or ZnPE1-Mb) as the negative control measurement (Fig. 4-16 to 4-17). Interestingly, even after the photoirradiation was stopped, the absorbance at 340 nm increased with time. These results showed that NADP⁺ was reduced not only photochemically but also chemically following photoirradiation of the solution.

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(A) (B)

(C) (D)

(E) (F)

Figure 4-14 UV-Vis spectral change for 10 μM ZnPP-Mb, 2.5 mM NADP⁺, and 1 M TEA in 100 mM KPi pH 9.0 under room temperature. (A) ZnPP-Mb^WT (B) ZnPP-Mb^H64D (C) ZnPP-Mb^V68L (D) ZnPP-Mb^I107M (E) ZnPP-Mb^H64D/V68L (F) ZnPP-MbH64D/V68L/I107M.

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(A) (B)

(C) (D)

(E) (F)

Figure 4-15 UV-Vis spectral change for 10 μM ZnPE1-Mb, 2.5 mM NADP⁺, and 1 M TEA in 100 mM KPi pH 9.0 under room temperature. (A) ZnPE1-Mb^WT (B) ZnPE1-Mb^H64D (C) ZnPE1-Mb^V68L (D) ZnPE1-Mb^I107M (E) ZnPE1-Mb^H64D/V68L (F) ZnPE1-MbH64D/V68L/I107M.

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(A) (B)

(C) (D)

(E) (F)

Figure 4-16 UV-Vis spectral change for 10 μM ZnPP-Mb, 2.5 mM NADP⁺, and 1 M TEA in 100 mM KPi pH 9.0 under room temperature and keep in dark for 5 days. (A) ZnPP-Mb^WT (B) ZnPP-Mb^H64D (C) ZnPP-Mb^V68L (D) ZnPP-Mb^I107M (E) ZnPP-Mb^H64D/V68L (F) ZnPP-MbH64D/V68L/I107M.

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(A) (B)

(C) (D)

(E) (F)

Figure 4-17 UV-Vis spectral change for 10 μM ZnPE1-Mb, 2.5 mM NADP⁺, and 1 M TEA in 100 mM KPi pH 9.0 under room temperature and keep in dark for 5 days. (A) ZnPE1-Mb^WT (B) ZnPE1-Mb^H64D (C) ZnPE1-Mb^V68L (D) ZnPE1-Mb^I107M (E) ZnPE1-Mb^H64D/V68L (F) ZnPE1-MbH64D/V68L/I107M.

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According to the report of Nishiyama et al.⁴⁰, to elucidate the mechanism of reduction, they used several kinds of reaction conditions and indicated that the photoirradiation of reconstituted metallo-porphyrin-Mb and TEA play an important role in the reduction. Furthermore, compared the results of photoirradiation and dark reaction (Fig. 4-16 to 4-17), it’s implied that photo-induction is necessary and controllable in the formation of NADPH. Fig. 4-18 and Table 4-3 to 4-4 showed the reduction rate of NADP⁺ in photoirradiation processwithin 6 hours. Compared the reaction rate between ZnPP-Mbs and ZnPE1-Mbs, ZnPE1-Mbs were faster (~0.19 mM/hr) than ZnPP-Mbs (~0.08 mM/hr). In addition, among various mutated myoglobins, not only reconstituted ZnPP-Mb^V68L but also ZnPE1-Mb^V68Lhad better reaction rate within 6 hours. To further study the relation between protein concentration and reaction rate within 6 hours, we used Mb^V68L with maximum reaction rate to do this analysis and compared to Mb^WT.

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(A)

(B)

Figure 4-18 The reduction rate of NADP⁺ in photoirradiation reaction within 6 hours. (A)ZnPP-Mb (B) ZnPE1-Mb.

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Table 4-3 Photoirradiation reaction of various ZnPP-Mb mutants on NADP⁺ reduction

Reaction rate in 6 hours (mM/hr)

ZnPP-Mb^WT 0.07064

Table 4-4 Photoirradiation reaction of various ZnPE1-Mb mutants on NADP⁺ reduction

Reaction rate in 6 hours (mM/hr)

ZnPE1-Mb^WT 0.19387

ZnPE1-Mb^H64D 0.18733

ZnPE1-Mb^V68L 0.20923

ZnPE1-Mb^I107M 0.1933

ZnPE1-Mb^H64D/V68L 0.20122

ZnPE1-MbH64D/V68L/I107M 0.19306

According to the results showed in Table 4-5, the reaction rate within 6 hours in ZnPP-Mbs increased when the concentration increased to 25 μM. However, these results did not happen in ZnPE1-Mbs. In higher concentration, the reaction rate decreased compared to 10 μM ZnPE1-Mb.

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Table 4-5 Reaction rate of metallo-porphyrins reconstituted with myoglobin within 6 hours

25μM (mM/hr) 10μM (mM/hr) 5μM (mM/hr)

ZnPP-Mb^WT 0.11525 0.07064 0.03684

ZnPP-Mb^V68L 0.13188 0.09132 0.03421

ZnPE1-Mb^WT 0.16479 0.19387 0.17598

ZnPE1-Mb^V68L 0.17182 0.20923 0.18215

Another interesting occurrence was observed in the photoirradiation prosess.

Before photoirradiation, a clear and sharp Soret band was observed at 428 nm for ZnPP-Mb and 443 nm for ZnPE1-Mb. Upon photoirradiation, there was an increase in the absorbance at 340 nm along with a decrease in the Soret band (Fig. 4-14 to 4-15).

Table 4-6 to 4-7 showed the change of Soret band in ZnPP-Mb and ZnPE1-Mb. There is a hypothesis about soret band decrease. The temperature increases upon photoirradiation process attribute to ZnPP-Mbs and ZnPE1-Mbs unthermostable.

Therefore, porphyrin is out of myoglobin result in Soret band decrease.

Table 4-6 The Soret band changed within 6 hours of ZnPP-Mb ΔSoret band (%)

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Table 4-7 The Soret band changed within 6 hours of ZnPE1-Mb ΔSoret band (%)

ZnPE1-MbH64D/V68L/I107M 70

Fig. 4-19 showed the results of 6 hours under photoirradiation and 5 days in the dark. At the fifth day, the increase at 340 nm became small extent and tended to equilibrium. Furthermore, compared conversion efficiency between ZnPP-Mbs and ZnPE1-Mbs, ZnPE1-Mbs had higher efficiency than ZnPP-Mbs. Table 4-8 to 4-9 summarized the results of NADPH formation for ZnPP-Mbs and ZnPE1-Mbs. Among various mutated myoglobins, not only reconstituted ZnPP-Mb^V68L but also ZnPE1-Mb^V68Lhad better conversion efficiency (Table 4-8 to 4-9). Comparing with the dark reaction, the NADPH formation was slight. It’s suggested that photo-induction is necessary in the formation of NADPH. In addition, according to these results, we deduced that metallo-porphyrin stabilization in myoglobin is not intense related to conversion efficiency. Upon photoirradiation, metallo-porphyrin-myoglobins became thermounstable resulting in Soret band decreased. The difference of conversion efficiency might be caused by the mutated amino acid, but the definite mechanism is not clear.

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(A)

(B)

Figure 4-19 Absorbance change at 340nm in the solution containing 10 μM ZnPE1-Mb, 1 M TEA, and 2.5 mM NADP⁺ in 100 mM phosphate buffer (pH 9.0).

(A) ZnPP-Mb (B) ZnPE1-Mb.

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Table 4-8 Conversion efficiency of ZnPP-Mb

Photoirradiation and dark reaction Dark reaction

ZnPP-Mb^WT 51.8 % 4.0 %

ZnPP-Mb^H64D 52.6 % 7.9 %

ZnPP-Mb^V68L 61.7 % 5.1 %

ZnPP-Mb^I107M 56.7 % 6.1 %

ZnPP-Mb^H64D/V68L 58.3 % 6.2 %

ZnPP-MbH64D/V68L/I107M 56.8 % 6.1 %

Table 4-9 Conversion efficiency of ZnPE1-Mb

Photoirradiation and dark reaction Dark reaction

ZnPE1-Mb^WT 72.7 % 9.8 %

ZnPE1-Mb^H64D 73.4 % 6.1 %

ZnPE1-Mb^V68L 94.7 % 6.2 %

ZnPE1-Mb^I107M 74.9 % 7.9 %

ZnPE1-Mb^H64D/V68L 92.6 % 9.4 %

ZnPE1-MbH64D/V68L/I107M 86.1 % 8.6 %

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