Part:BBa_K2346000

Nitric oxide reductase (NOR)

Cytochrome P450 55B1 from Chlamydomonas reinhardtii is reported to function as a nitric oxide reductase (NOR). We are still working on that. Since nitric oxide is nearly insoluable in water, it’s hard for us to create a aquatic reaction system for assaying the enzyme reactivity. We are still working on that. But currently we plan to create a reaction environment in a sealed container filled with solution, enzyme, and nitric oxide gas, By constant shake over, let’s say, two hours, we’ll then measure the change in the concentration of NO using the Gas-Phase Molecular Absorption Spectrometry to see if the presence of the enzyme made any difference on the concentration.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1
Illegal XhoI site found at 1216
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1200
1000
COMPATIBLE WITH RFC[1000]

Background

Cytochrome P450 55B1 from Chlamydomonas reinhardtii
General Equation: $ \ce{2NO + NAD(P)H + H+ → N2O + NADP+ + H2O} $

We found three assay methods to test the activity of nitric oxide reductase: using NO measurement Kit, titration and Gas-Phase Molecular Absorption Spectrometry. The first two methods were failed, and the details and reasons were recorded below. Due to limitations of lab equipment, we were not able to conduct the experiment using the third method.

Nitric oxide reductase's tertiary structure

Assay method 1: NO Measurement Kit

The NO kit we planned to use was developed by Nanjing Jiancheng Bioengineering Institute, and the concentration of NO in the solution can be calculated by the concentration of $\ce{NO3-}$, because NO will be reduced to $\ce{NO3-}$ in blood plasma of chicken or mice. We prepared the solution according to the method as below:

1	Remove the air in the flask by passing through nitrogen gas for 30 minutes.
2	Pass through nitric oxide gas for 45 minutes in Flask C that contains ddH2O.
3	Flask A contains 30% H2O2 solution, and Flask B contains KMnO4 / NaOH solution, to oxidize and absorb the undissolved gas.[1]

However, although the NO solution we prepared using this method was saturated, the concentration was too low for the verification to proceed.

Fig 1. Setup of equipments for preparing NO solution

Fig 2. NO gas container

Fig 3. Equipments setup

Assay Method 2: Titration

By measuring the concentration of nitrous oxide—the final product of the reduction of nitric oxide, the amount of nitric oxide reacted can be calculated, and therefore the activity of nitric reductase can be verified. When the reaction is complete, acidic potassium permanganate is added to the solution, and the highly oxidative potassium permanganate reacts with nitrous oxide. We then use oxalic acid to titrate the remaining potassium permanganate. Therefore, the amount of potassium permanganate used in the reaction with nitrous oxide can be calculated, and the amount of nitrous oxide produce can be found.

However, even for the most stable solution of nitrous oxide—the standard solution of 1.00mg/ml, nitrous oxide will escape in gas form, and the escaping speed is not constant. As a result, the method of directly measuring the amount of nitrous oxide is not valid.

Fig 4. Standard N2O solution

Assay Method 3: Gas-Phase Molecular Absorption Spectrometry

The turnover of the overall reaction of nitric oxide reductase was determined by monitoring the NADH consumption rate under various concentrations of NO, where the NO concentration was controlled by bubbling the NO/N2 mixed gas produced with the gas divider (ESTEC SGD-SC-0.5L). Unfortunately, since we could not find access to the required gas divider, we were not able to verify the results. The results from the original paper was shown in Fig 4.

Protocol

1	Prepare the buffer: 0.1 M sodium phosphate at pH 7.2.
2	Add reduced form of NADH.
3	Purge the air in the buffer solution by nitrogen gas to prevent formation of NO2 before adding NO.
4	Introduce NO gas into the sample solution after passing 1 M KOH and the buffer solutions.
5	Incubate the buffer solution containing NO (2.5, 2.0, 1.6, 1.5, 1.2, or 1.0 mM) and NADH (0.16 mM) in the optical cell at 10°C for 5 min.
6	Measure the absorbance (340nm) of the solutions.

Results

Due to limited access to equipments, we could not directly measure the reactivity of NOR. However, experiments had been carried out for the same enzyme expressed also expressed in E.coli, with method listed above. For the continuation of our project, we had to draw conclusion from other’s experiment results.

Fig 5. Time course of the absorption change of the reduced form of NADH at 340 nm in the NO reduction reaction catalyzed by P450nor. [2]

Conclusion

We delightedly found out that the curve became smooth and parallel to the x-axis just within the course of five minutes. This represented nearly all the substrates had been converted to products in an extremely rapid rate: much more rapid than nitrite reductase. This means that whenever NO is released from nitrite reductase as product, NOR can utilize it as substrate quickly.

Reference

1	程得胜, 黄曜, 黄郁芳. 一氧化氮标准储备水溶液浓度的测定及稳定性分析[J]. 分析测试学报, 2007, 26(4):566-569.
2	Shiro Y., Fujii M., Iizuka T., Adachi S., Tsukamoto K., Nakahara K., Shoun H. 1995. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. J. Biol. Chem. 270, 1617–162310.1074/jbc.270.4.1617.
3	Shoun H, Fushinobu S, Jiang L, et al. Fungal denitrification and nitric oxide reductase cytochrome P450nor[J]. Philosophical Transactions of the Royal Society of London, 2012, 367(1593):1186.

Background

The first enzyme NiR is the product from expression of nirS gene amplified from Alcaligenes eutrophus DSM 530. The NiR catalyzes the reduction of nitrite (NO2-) to nitrogen monoxide(NO), as the first step of our substrate channeling system.

Biology

Nitrite reductase's tertiary structure

We expressed the nitrite reductase with a His-tag in E.coli using a pET28a vector. The proteins are then extracted from the cells and purified using a HIS-trap column. After further purification, we ran electrophoresis to check the correct expression of NiR. The result of protein electrophoresis is shown below.

Assay method

We used N-(1-naphthyl)ethylene diamine dihydrochloride spectrophotometric method, a common approach in waste water treatment, to measure the concentration of nitrite in the solution to track the reaction process. The nitrite concentration was measured using a color-showing reagent consisting of p-aminobenzenesulfonamide and 1g n-(1-naphthyl)-ethylenediamine dihydrochloride.

The reagent reacts with nitrite to form the redish pink diazo salt in the solution, which has a maximum absorbance around 540nm. A standard curve was first made by measuring the absorbance at 540nm of the reaction system under various nitrite concentration.

Stand curve between Absorbance-540nm and Sodium Nitrite concentration is shown below.

Standard curve between Absorbance-540nm and Sodium Nitrite concentration

Reactivity assay in vitro

To assay the reactivity of the protein. We put the enzyme, sodium nitrite and pbs buffer together to establish a reaction system. We recorded the nitrite concentration over 24 hours, under temperature of 37 degree Celcius. A standard curve was first made(as seen above) by measuring the absorbance at 540nm of the reaction system under various nitrite concentration.

According to our results, the absorbance dropped from 0.633 to 0.008 over 24 hours, which indicates a drop of nitrite concentration from 0.196 mg/L to roughly zero. Currently we are still testing samples with shorter and various reaction time-length to plot the closest curve of the reactivity.

NiR reactivity assay in vitro

Since the application of these enzymes will be in vivo, we are satisfied with NiR’s reactivity was verified. We are much more interested in NiR’s ability to react in vivo with much greater concentration of nitrite.

Reactivity assay in vivo

Protocols

	Add IPTG into prepared cultures of E.coli (IPTG concentration: 0.1mg/L)
	Induce at 16 °C, 150 rpm overnight
	Add sodium nitrite solution at various concentration (Target concentration: ~65mg/L)
	Culture the bacteria at 37 °C,150 rpm
	Take samples (500uL) of the cultures at various time points
	Measure optical density(600nm) of the samples
	Centrifugate at 6,000 rpm for 60s
	Take 100uL samples from supernatant and dilute with 900uL ddH2O
	Take 100uL samples from the diluted solution and dilute again with 900 uL ddH2O
	Add 20uL of chromogenic agent to the solution
	Stand the solution for 20 mins and wait for the color to develop.
	Measure the absorbance(540nm) of the solutions
	Record data in Excel forms

*Prepare the chromogenic agent as follows:

1

In 500mL beaker <tbody> </tbody>

	50mL Phosphoric Acid (d=1.70g/m)
	20.0g p-aminobenzenesulfonamide (C6H8N2O2S)
	1g n-(1-naphthyl)-ethylenediamine dihydrochloride
	250mL ddH2O

2

Transfer the solution to a 500mL volumetric flask and dilute with ddH2O to standard volume.

NOTICE: We diluted the solution x100, so you have to time 100 the corresponding nitrite concentration using standard curve.

Results

Nitrite conc. against reaction time

	According to our results, the absorbance at 540nm dropped from 0.410 to 90.105 over 45.5 hours, which indicates a drop of NaNO2 concentration from 62.0(mg/L) to 14.9(mg/L).
	According to the graph, the reactivity of NiR is the highest during 20-30hours. About 76% of total nitrite is degraded at the end of assay. Also, there is no obvious reduction in nitrite concentration in control group.