Direct Spectroscopic Ferrochelatase Assay

Abstract

Ferrochelatases (E.C. 4.99.1.1) catalyzes the insertion of ferrous iron into either protoporphyrin IX to make protoheme IX or coproporphyrin III to make coproheme III. Ferrochelatase activity in extracts or purified protein can be measured via several assays. Herein we describe a rapid real-time direct spectroscopic ferrochelatase assay for both protoporphyrin and coproporphyrin ferrochelatases.

1. Introduction

Ferrochelatases (E.C. 4.99.1.1) are enzyme which catalyzes the insertion of ferrous iron into porphyrin macrocycles. While ferrous iron is the physiological metal substrate for all ferrochelatases, there are different types of ferrochelatases that utilize different porphyrins as substrates, either protoporphyrin IX or coproporphyrin III [1]. The protoporphyrin ferrochelatases are part of the protoporphyrin dependent (PPD) heme synthesis pathway found in proteobacteria and eukaryotes. Protoporphyrin ferrochelatases catalyze the terminal step in the protoporphyrin dependent heme synthesis pathway to produce protoheme IX and two proton. The coproporphyrin ferrochelatases are part of the coproporphyrin dependent (CPD) pathway which is found in most diderm bacteria in the Firmicutes and Actinobacteria Phyla. Coproporphyrin ferrochelatases catalyze the penultimate step of the CDP pathway to produce coproheme III which is then decarboxylated to protoheme IX. The CDP pathway was not discovered until 2015 [2], thus it is important to note that all studies prior to this time incorrectly assumed protoporphyrin IX was the substrate of all ferrochelatases.

There are a number of assay procedures that have been used to evaluate ferrochelatase activity and determine kinetic parameters. Two of these assays are discontinuous and quantitate product either as its oxidized minus reduced pyridine hemochromagen [3,4] or via radioactive iron incorporation [5]. The radioactive assay, while most sensitive, is seldom utilized due to its arduous nature and issues associated with obtaining ⁵⁹Fe or ⁵⁵Fe. The continuous assay for ferrochelatase activity is based upon changes in the spectrum of free porphyrin versus metalated porphyrin. The direct spectroscopic assay uses selected wavelength-specific absorbance, zeroed to absorbance at isosbestic points to measure in real-time the disappearance of porphyrin substrate or appearance of metalated product [2,6,7]. Additionally, one may follow the loss of fluorescence of the free porphyrin as its converted to the nonfluorescent metalated porphyrin [8]. While quantitation of product by pyridine hemochromagen is more sensitive than the direct spectroscopic determination of product formation or substrate disappearance, the continuous assay is now the one most commonly employed since it is more rapid and avoids the use of pyridine and the need for a split beam spectrophotometer. Herein we summarize details of the real-time direct spectroscopic ferrochelatase assay and its use for both protoporphyrin and coproporphyrin ferrochelatases.

2. Materials

Prepare all solutions using ultrapure water at room temperature.

Assay Reagents:

1. Ferrochelatase assay buffer: 100 mM Tris-HCl, pH 8.0 and 0.5% Tween 20 (see Note 1). For 100 mL of assay buffer 10 mL of 1M Tris-HCl, pH 8.0, 5 mL of Tween 20 Surfact-Amps Detergent 10% Solution, and 85 mL of ultrapure water are mixed.

2. Reductant: 100 mM β-mercaptoethanol (see Note 2). A stock solution of 100 mM β-mercaptoethanol is prepared by diluting 70 microliters of β-mercaptoethanol in 10 mL of ultra-pure water.

3. Porphyrin stock solution: 1 mM porphyrin. Porphyrin stock solutions should be freshly prepared and stored in the dark and cold. Use only glass tubes which should be darkly colored (black or brown) or covered in aluminum foil to protect from light. Do not use polypropylene or polystyrene, as porphyrins stick to these. To make ~1 mM solution of porphyrins, measure out between 2.5 to 4 mg of porphyrins (i.e. mesoporphyrin IX, protoporphyrin IX, deuteroporphyrin IX, coproporphyrin III, etc.). Add 50 μl of concentrated ammonium hydroxide. Vortex well to make a paste. Add 500 μl of 10% Triton X-100 and mix until there is a clear solution, i.e. no undissolved porphyrin. Add 5 mL of ultrapure water and vortex to mix (see Note 3).

4. Iron stock solution: 1 mM ferrous ammonium sulfate. For metals, use a 15 mL conical tube, either polypropylene or polystyrene. To make a ~1 mM solution of iron, weigh out 2 mg of ferrous ammonium sulfate. Add 5 mL of ultrapure water and vortex to mix. Add a small amount (a crystal or pinch) of ascorbic acid. Iron solutions should be freshly prepared (see Note 4).

3. Methods

Carry out all assays at a temperature appropriate to the source of enzyme. This may be room temperature or in a thermostated cuvette. Assay solutions should be held at the appropriate temperature prior to initiation of the assay to avoid warming during the assay period. Mesoporphyrin IX or deuteroporphyrin IX are used except for kinetic measurements since those two porphyrins are more soluble and stable in solution than protoporphyrin IX. Coproporphyrin III, which is used only for coproporphyrin ferrochelatases, is similar in properties to deuteroporphyrin IX and mesoporphyrin IX with regard to its solubility. Early ferrochelatase assays were frequently run under anaerobic conditions, but this has been shown not to be a necessity.

Porphyrin and Iron Quantitation

1. To quantitate porphyrins, dilute porphyrin 1:1000 in a glass tube in the appropriate concentration of HCl and scan from 300 to 500 nm (see Note 5).

2. To quantitate the iron, dilute iron 1:100 in in a conical tube. Add 100 μl 10 mM ferrozine solution. Measure the absorbance of 1 mL of the iron:ferrozine solution at 562 nm and use an extinction coefficient of 0.278 mM⁻¹ (see Note 6).

Assay

1. Add the following to a cuvette and mix

   900 μl of assay buffer

   100 μl of 100 mM β-mercaptoethanol (or 50 mM dithiothreitol)

   100 μl of 1 mM of selected porphyrin

   100 μl of 1 mM ferrous ammonium sulfate (or other selected metal)

   Insert cuvette into spectrophotometer.

2. Add 10 μl of ~100 μM purified ferrochelatase or an appropriate amount of cell extract to allow one to follow changes in the observed spectrum.

   Mix rapidly with a sealed, bent capillary tube and start recording absorbance immediately (see Note 7). For purified enzyme, scans covering the appropriate wavelengths should be run every 30 seconds for approximately 3 min. For less active preparations longer times may be required. However, one should avoid leaving the assay cuvette in the light beam for excessive lengths of time since porphyrins are light sensitive and will degrade.

3. For assays in which the disappearance of substrate is being measured, the slope will be negative.

4. For kinetic measurements, change the amount of one substate while holding the other constant (see Note 8).

4. Notes

1. The Tween 20 Surfact-Amps Detergent Solution is a highly-purified Tween-20 detergent stabilized and supplied as a 10% solution in 10mL glass. While Tris-HCl is employed herein, any buffer that functions at pH 7.5 to 8.0 is acceptable.

2. A reductant is also included in all assay buffer to maintain the iron ion in a reduced state. This reductant may be either dithiothreitol or β-mercaptoethanol. For either, solutions are freshly prepared.

3. If measuring kinetic parameters, porphyrin solutions should be used immediately since porphyrins will stack and precipitate out of aqueous solution. For routine assays, stock solutions of mesoporphyrin IX, deuteroporphyrin IX or coproporphyrin III may be stored overnight in the cold. Protoporphyrin should always be freshly prepared.

4. Other metals, such as Zn, Co, and Ni, may be prepared in advance and stored at room temperature.

5. For different porphyrins the concentration of the HCl and extinction coefficient differs. A full list can be found here [9] and specific porphyrins are described herein. For protoporphyrin IX dilute in 2.7 N HCl and use extinction coefficient of 13.5 mM⁻¹ at 554 nm. For coproporphyrin III dilute in 0.1 N HCl and use extinction coefficient 16.8 mM⁻¹ at 548 nm. For mesoporphyrin IX dilute in 0.1 N HCl and use the extinction coefficient 15.1 mM⁻¹ at 547 nm and for deuteroporphyrin IX dilute in 0.1 N HCl and use extinction coefficient of 13.7mM⁻¹ at 548 nm.

6. For other divalent metals such at nickel, copper, cobalt and zinc. Make a 10 mM stock and dilute down to 1 mM for assays. For routine assays we frequently employ Ni and follow product formation since it avoids the problem of metal oxidation that one has with ferrous iron. Ni-porphyrin yields a product whose absorbance maximum is well shifted from free porphyrin. Zn insertion is employed by some since one can follow the product formation using fluorescence. However, Zn-porphyrin is considerably less stable than other metals. Early continuous assays employed Co [6], but for some ferrochelatases Co is less active than other metals.

7. The isosbestic point for each metal with each porphyrin differs [2]. Wavelength pairs are: deuteroheme IX, 496–484, Δε_mM = 3.5, mesoheme IX 497–486, Δε_mM = 7.5, protoheme, 497–486, Δε_mM = 10.5. Alternatively one may follow protoporphyrin disappearance at 385 nM (58 mM⁻¹cm⁻¹) [10], and coproporphyrin disappearance at 385 (128 mM⁻¹cm⁻¹) [11]. For Ni insertion into mesoporphyrin IX, use nickel chloride and follow an increase in absorbance at 550 nm. Scan assay mixture every 30 seconds for 3 minutes.

8. Appropriate volume of variable substrate to use for kinetics are 100, 80, 60, 50, 40, 30, 25, 20, 15, 10 μl. Perform all assays in triplicate.