Class Ib Ribonucleotide Reductases: Activation of a Peroxido-MnIIMnIII to Generate a Reactive Oxo-MnIIIMnIV Oxidant

In the postulated catalytic cycle of class Ib Mn2 ribonucleotide reductases (RNRs), a MnII2 core is suggested to react with superoxide (O2·–) to generate peroxido-MnIIMnIII and oxo-MnIIIMnIV entities prior to proton-coupled electron transfer (PCET) oxidation of tyrosine. There is limited experimental support for this mechanism. We demonstrate that [MnII2(BPMP)(OAc)2](ClO4) (1, HBPMP = 2,6-bis[(bis(2 pyridylmethyl)amino)methyl]-4-methylphenol) was converted to peroxido-MnIIMnIII (2) in the presence of superoxide anion that converted to (μ-O)(μ-OH)MnIIIMnIV (3) via the addition of an H+-donor (p-TsOH) or (μ-O)2MnIIIMnIV (4) upon warming to room temperature. The physical properties of 3 and 4 were probed using UV–vis, EPR, X-ray absorption, and IR spectroscopies and mass spectrometry. Compounds 3 and 4 were capable of phenol oxidation to yield a phenoxyl radical via a concerted PCET oxidation, supporting the proposed mechanism of tyrosyl radical cofactor generation in RNRs. The synthetic models demonstrate that the postulated O2/Mn2/tyrosine activation mechanism in class Ib Mn2 RNRs is plausible and provides spectral insights into intermediates currently elusive in the native enzyme.


Cautions:
Perchlorate salts of metal complexes and KO 2 are potentially explosive and must be handled with care and in small quantities.We limited our preparations of 1 to a maximum of 0.2 g per reaction.

WARNING -PREPARATION AND STORAGE OF K 18 O 2 :
We had a minor explosion, inside our glovebox, when handling (using a metal spatula) a small quantity (20-40 mg) of our in-house prepared K 18 O 2 in a glass Schlenk flask.There was no spark or initial flame, the ignition was rapid and forceful.The explosion caused the Schlenk flask (volume = 20 mL) to shatter, but no injury to the researcher.We believe injury was avoided because the explosion occurred inside the glovebox, while the researcher was protected from the shattering glass by the glovebox structure and heavy-duty gloves.A more serious outcome was likely had the explosion not been inside the glovebox.The water content in the glovebox was below detectable levels at the time.We tentatively postulate that the sample contained some K Matlab and the easySpin computational package. 6ray absorption spectroscopy (XAS) methods: The Mn-K-edge X-ray absorption data were collected on beamline 7-3 of the Stanford Synchrotron Radiation Lightsource (SLAC National Accelerator Lab, Menlo Park, CA, USA).Data were collected with the storage ring operating at 3.0 GeV and 500 mA, using a LN2 cooled Si(220), ϕ = 90˚ double-crystal monochromator, calibrated by using the first inflection point of a Mn foil (6539.0eV).The monochromator was detuned by ~50% for higher harmonic rejection.A Canberra 32-element solid state germanium detector was used for fluorescence detection.All measurements were performed at ambient pressure at ~17 K using an Oxford helium cryostat that was cooled by closed-cycle He gas loop.The parameters used for the scans were the following: 10 eV steps/1 second integration time in the pre-edge region, 0.3 eV steps /2 second integration time in the edge, and 0.05k steps above the edge, with integration time increasing in a k 2 -weighted fashion from 2 to 4 seconds over the selected energy range (k max = 12.1k).The total detector counts were typically 3-7k, well within the linear range of the detector electronics.Each sample was monitored for photoreduction.Complexes 2, 3, and 4 were found to be photoreduced by the X-rays and so only one scan was obtained per spot.Evaluation of the XAS data, including averaging, background removal and normalization, was performed using Athena.7 Edge energies were obtained by taking the first derivative of the rising edge, while pre-edge energies were identified using the second derivative.

Experimental Procedures
General procedure for the preparation of 2: A CH 3 CN/THF (1:9) solution of 1 (1.5 mM) was prepared.In a quartz cuvette, 2 mL of this solution was cooled to -90 °C.KO 2 (0.0056 g, 0.04 M) and cryptand (0.06 g, 0.08 M) were dissolved in DMF (2 mL).75 L of this solution was added to the solution containing 1.The  reaction progress was monitored using electronic absorption spectroscopy.]

S5
General procedure for the preparation of 3: 2 was prepared as above.para-Toluene sulfonic acid (p-TsOH) was dissolved in 1:9 CH 3 CN/THF (0.095g, 0.55 M).This solution was added to the solution containing 2 (volume depended on the stoichiometry required).The reaction progress was monitored using electronic absorption spectroscopy.
General procedure for the preparation of 4: 2 was prepared as above at -90 °C.The temperature controller on the Unisoku cryostat was subsequently programmed to 20 °C resulting in the gradual warming of the solution to 20 °C.
The reaction progress was monitored using electronic absorption spectroscopy.

Synthesis of ABNO-H
ABNO-H was synthesised used an adapted procedure. 11Na 2 S 2 O 4 (1.00 g, 5.7 mmol) was added to a degassed solution of ABNO (500 mg, 3.7 mmol) dissolved in 1:1 acetone/water (15 ml) resulting in a colour change from orange to white.The acetone was removed under vacuum and the resulting aqueous solution was extracted with deoxygenated pentane (3 x 10 ml).The pentane was removed under vacuum to yield a white precipitate and triturated with ~15 ml Et 2 O followed by solvent removal under vacuum (yield = 342 mg, 70%).The 1 H NMR spectrum was consistent with previously published spectra of ABNO-H. 12lculation of ABNO-H BDFE in CH 3 CN 13,14 : ∆°  (°) = 5.11   -1

EPR sample preparation:
Samples for EPR analysis were prepared by transferring ~ 1 mL of the desired solution from the quartz cuvette at -90 °C, via a pre-cooled pipette into a pre-cooled EPR tube and immediately freezing it in liquid nitrogen.The EPR spectra were recorded at 2 K (9.64 GHz, 0.2 mW microwave power).

ESI-MS sample preparation:
The samples for frozen MS analysis were prepared by transferring ~ 0.1 mL of the desired solution from the quartz cuvette, pre-cooled to -90 °C, via a pre-cooled sample vial and immediately freezing in liquid nitrogen.ESI-MS was performed on the just thawed sample.
Once the sample had started to melt it was taken into a pre-cooled syringe and rapidly injected directly into our micromass time of flight mass spectrometry instrument.The spray-head temperature was 180 °C and the accelerating voltage was 4,000 V.

Reactivity Studies:
3 and 4 were prepared as described above.Substrates were added as concentrated CH 3 CN/THF solutions to solutions of 3 at -90 °C or 4 at +20 °C.The reactions were monitored using electronic absorption spectroscopy.

Note on the Hill
Type kinetics observed in the reaction of 4 and 4-CN-2,6-DTBP: system displaying non-linear k obs vs [S] (S = substrate) behaviour may be understood as more than one substrate molecule is involved in the reaction.The cooperativity for the reaction between 4 and 4-CN-2,6-DTBP was calculated to be 2.95 1.The change from Michaelis-± Menten behaviour to a Hill type may be attributed to an increase in acidity of the O-H bond of 4-CN-2,6-DTBP.

Figure S1 .
Figure S1.Electronic absorption spectra of 2 generated from the reaction of 1 (1.5 mM) with

Figure S4 .
Figure S4.Perpendicular mode X-Band EPR spectra of the post-reaction mixture of 2 (black)

Figure S7 .
Figure S7.Left and centre: ESI-MS spectra of 3 prepared using K 16 O 2 (black trace) with

Figure S8 .
Figure S8.ESI-MS of sample containing 3 expanded in the regions where 2 is found.

Figure S17 .
Figure S17.Post-reaction electronic absorption spectrum from the reaction of 3 with 4-CH 3 O-

Figure S18 .
Figure S18.Electronic absorption spectra changes during the reaction of 3 (black trace) with

Figure S19 .
Figure S19.Electronic absorption spectra changes during the reaction of 3 (black trace) with

Figure S20 .Figure S21 .
Figure S20.Electronic absorption spectra changes during the reaction of 3 (black trace) with

Figure S22 .
Figure S22.Plot of the absorbance at  = 550 nm during the reaction of 3 and ABNO-H (black

Figure S24 .
Figure S24.Plot of the absorbance at  = 550 nm during the reaction of 3 and 4-CH 3 O-

Figure S27 .
Figure S27.Plot of k obs versus [S] determined for the reaction of 3 and ABNO-H.

Figure S28 .
Figure S28.Plot of k obs versus [S] determined for the reaction of 3 and TEMPO-H.

Figure S29 .
Figure S29.Plot of k obs versus [S] determined for the reaction of 3 and 4-CH 3 O-TEMPO-H.

Figure S30 .
Figure S30.Plot of k obs versus [S] determined for the reaction of 3 and 4-oxo-TEMPO-H.

Figure S31 .
Figure S31.EPR spectrum from the reaction of 3 with ABNO-H.

Figure S32 .
Figure S32.EPR spectrum from the reaction of 3 with TEMPO-H

Figure S36 .Figure S37 .
Figure S36.Plot of ΔG ‡ versus the BDFE O-H values for the reactions between 3 and ABNO-

Figure S39 .
Figure S39.Electronic absorption spectra changes during the reaction of 4 (black trace) with

Figure S40 .
Figure S40.Electronic absorption spectra changes during the reaction of 4 (black trace) with

Figure S41 .
Figure S41.Electronic absorption spectra changes during the reaction of 4 (black trace) with

Figure S42 .
Figure S42.Electronic absorption spectra changes during the reaction of 4 (black trace) with

Figure S43 .
Figure S43.Electronic absorption spectra changes during the reaction of 4 (black trace) with

Figure S47 .
Figure S47.Plot of the absorbance at  = 590 nm during the reaction of 4 and 2,6-DTBP (black

Figure S60 .
Figure S60.EPR spectrum of the post-reaction mixture from the reaction of 4 with 2,4,6-

Figure S61 .
Figure S61.EPR spectrum of the post-reaction mixture from the reaction of 4 with 2,6-DTBP.

Figure S62 :
Figure S62: Plot of ΔG ‡ versus the BDFE O-H values for the substrates.Values of ΔG ‡ were

Figure S63 .
Figure S63.Plot of the log(k 1 ) versus pK a of the phenol substrates for 4.

Figure S64 .
Figure S64.Plot of log(K M ) versus pK a of the phenol substrates for 4.

Figure S65 .
Figure S65.Plot of (RT/F)ln(K M ) versus E OX for the phenol substrates for 4.

Figure S66 .
Figure S66.Plot of (RT/F)ln(k 1 ) versus E OX for the phenol substrates for 4.

Figure S68 .
Figure S68.Plot of 1/k obs versus 1/[H-4-CH 3 O-2,6-DTBP] determined for the reaction between 5 O 2 .K 2 O 2 is considerably more shock-sensitive than KO 2 .As a result of this we have decided to not use/store/prepare K 18 O 2 locally again.The method of K 18 O 2 preparation was from a previously reported procedure, reacting TEMPO-K with 18 O 2 .5 cuvettes on a Hewlett Packard (Agilent) 8453 diode array spectrophotometer (190 -1100 nm range) coupled to a liquid nitrogen cooled cryostat from Unisoku Scientific Instruments (Osaka, Japan).Electron paramagnetic resonance (EPR) measurements were conducted on a Bruker Elexsys E-500 spectrometer with an Oxford ESR 910 liquid helium cryostat and an Oxford temperature controller.The quantification of EPR signals measured at 30 K was against a [Cu II (NO 3 ) 2 ] spin standard in CH 3 OH.The quantification of EPR signals measured at 77 K were relative to a TEMPO spin standard in 1:9 CH 3 CN/THF.EPR spectra of 4-X-TEMPO and ABNO were