Catalytic and Spectroscopic Properties of the Halotolerant Soluble Methane Monooxygenase Reductase from Methylomonas methanica MC09

Abstract The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2 M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.


Plasmid construction
For heterologous overproduction and subsequent purification, the mmoC gene from Methylomonas methanica MC09 was codon optimised for E. coli and inserted via the NdeI and BamHI restriction sites in the plasmid pET-16b. The 5´ end of mmoC was equipped with a 10xHis-tag-encoding sequence resulting in the plasmid pZD02 (Fig. S1).

MmoC production and purification
E . coli BL21 with the plasmids pBB540 + pBB550 for the co-production of the chaperons DnaK, DnaJ, GrpE, ClpB, GroESL and pZD02 were grown in rich Terrific Broth (TB) medium at 37 °C until OD600nm of 2 and were then induced with 0.1 mM IPTG. The protein production phase was performed at 18 °C for 14 h. The harvested cells were resuspended in twice their volume of resuspension buffer (50 mM K-PO4, pH 7.2, 500 mM NaCl containing additional Protease Inhibitor (EDTA-free, Roche) and DNase I). After two passages through a chilled French pressure cell at 6.2 MPa, the suspension was centrifuged at 100,000× g for 45 min. The soluble extract was applied to a 2 mL Ni-NTA affinity chromatography column, washed with 6 mL of resuspension buffer without protease inhibitor and DNase I and eluted with the same buffer containing 500m mM imidazole. The eluate was then concentrated in an Amicon Ultra-15 centrifugal cell (10 kDa membrane; Amicon, Witten, Germany). We isolated in average 16.2 mg (average of in total four purifications) homogenous MmoC from 1 g cell pellet (wet weight). In order to monitor the purification, samples of every purification step were analysed by SDS-PAGE ( Fig  S2). In the elution fraction, a pronounced band was detected, which correspond to the MmoC calculated size of 37.9 kDa. A second thin band of around 70 kDa most likely correspond to the co-produced chaperon DnaK. [1] Because of the already high purity of MmoC (>95%), we decided to not proceed with further purification steps. The pronounced MmoC band in the cell extract in comparison to the soluble extract indicated that a part of the produced MmoC is accumulated in inclusion bodies. The yield of proteins resulting in inclusion bodies could be increased by adding an in vitro refolding step to the purification protocol of E. coli BL21 containing pBB540 + pBB550 [2] . As we already achieved high yields with our existing protocol, we did not apply an in vitro refolding step to the purification protocol. [1] To remove imidazole after Ni-NTA affinity chromatography, the elution buffer of MmoC was exchanged via an Amicon Ultra-15 centrifugal filter (10 kDa membrane) to 50 mM potassium phosphate buffer pH 7.5 with 5% glycerol. Protein concentrations were determined with BCA protein assay kit (Pierce, USA) as described previously [3] .

Spectroscopic measurements
Samples' UV/visible spectra were recorded with a Varian Cary 300 instrument at 16°C. The final working concentration of protein samples was 26 µM. The MmoC was reduced with 2 mM sodium dithionite under anaerobic conditions. Electron Paramagnetic Resonance (EPR) spectroscopic experiments were performed on a Brucker EMX plus X-Band spectrometer equipped with an ER 4122 super-high Q (SHQE) resonator (Bruker Corporation) and an Oxford ESR900 helium flow cryostat (Oxford Instruments). During the measurements, an Oxford ITC4 (Oxford Instruments) temperature controller was used for adjusting the temperature. Baseline correction was performed by the subtraction of a reference spectrum obtained from a sample of buffer solution recorded with the same experimental parameters. For subsequent corrections a polynomial or spline function was used. Experimental parameters used was: 1 mW microwave power, microwave frequency 9.29 GHz, modulation amplitude 10 G and 100 kHz modulation frequency. The oxidized and NADH-reduced MmoC samples for EPR spectroscopy were analysed with a concentration of 50 µM in a volume of 100 µL.

Determination of FAD saturation
Flavin adenin dinucleotide (FAD) concentrations in MmoC were analysed photometrically in a plate reader (SpectraMax 340PC-384) at 450 nm wavelength as described previously. [4] Briefly, protein samples (10 and 3 mg/mL) were denatured by mixing with 20% trichloroacetic acid (TCA) in 1:1 ratio for 10 min at 4°C. After precipitation, the mixture was neutralised with half of the volume of the protein samples. The maintained solution was used for double determination and was filled up to 200 µL. FAD (95%) was used to prepare standards.

Optical emission spectroscopy
For the determination of iron in MmoC, metal analysis was performed using a Perkin-Elmer Optima 2100DV inductively coupled plasma-optical emission spectrometer (Perkin-Elmer, Fremont, CA, USA) following the protocol described previously [5] . In short, 500 µL of protein samples were incubated overnight with equal amount of 65% nitric acid (Suprapur, Merck KGaA, Darmstadt, Germany) at 100 °C. Samples were filled up to 5 mL with water prior to ICP-OES analysis. Buffer samples without protein were treated the same way to check if footprint of metal is dissolved in the buffer. As reference, the multielement standard solution XVI (Merck) was used.

Activity measurements
The NADH oxidation activity was measured under anaerobic conditions in a N2-saturated activity buffer (50 mM K-PO4 buffer pH 7.2, 0.25 M NaCl, 5 mM benzyl viologen, 1 mM NADH, ca. 100 µM sodium dithionite) similar to published literature [4] . The reaction was started by adding 2.5 µL purified MmoC (0,1 µM) to the buffer, and reduction of benzyl viologen was followed at 578 nm (VARIAN Cary 50 BIO UV-Visible Spectrometer, ε=8.9 mM-1 cm-1). For the determination of reaction optima, first of all, the salt and then the pH optima were determined at 20 °C (RT). At the end, the determination of the temperature optimum was performed. Salt concentrations were analysed in range of 0-2 M NaCl (due to marine habitat from M. methylomonas), pH values ranged from 6.0-8.5, and temperature studies were performed from 10-50 °C. Each measurement was performed with technical triplicates. Activity determination of MmoC with added FAD/ FMN for reconstitution investigations were measured with a biological replicate but also with technical triplicates.

Peroxide detection assay
A spectrophotometric assay previously described by Fredrico et al 1997. [6] was adopted and modified for the detection of hydrogen peroxide produced by MmoC. The assay is described as follows: A reaction mixture of 5.5 µL of NADH (final concentration 275 µM), 2.5-3.0 µL of purified MmoC (final concentration 2 µM) and 1 mL of 50 mM Tris-HCl buffer pH 7.2 was prepared in 1.5 mL Eppendorf tube. This reaction mixture was incubated at 30 °C for up to 5 min in a shaking thermoblock. The reaction was stopped by adding 1 ml of 3% trichloroacetic acid. The reaction mixture was centrifuged at 13,000xg for 5 min and supernatant was transferred to a new tube which was further neutralized with 5 M NaOH to pH 7.4-8. The colorless neutral solution (2 mL) was transferred to a 5 mL glass cuvette. Then, 50 µL each of AAP (final concentration 0.5 mM) and DCHBS (final concentration 5 mM), and 3 µL HRP (final concentration 4 U/mL) were added and properly mixed by inverting the cuvette three times. The formation of the pink dye was monitored spectroscopically at 515 nm (ε515 = 26000 M -1 *cm -1 ) using Cary 50 (Varian). A negative control without HRP was used. For H2O2-concentration determination, a calibration line was plotted with the known amount of hydrogen peroxide (range used between 0 to 80 µM).