Hydrogen production by a fully de novo enzyme

Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterised by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo enzyme produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with turnover numbers around 80% of that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, how our study contributes important information about the functionality of cobaloximes as hydrogen evolving catalysts in protein environments, and the feasibility of using de novo proteins for development into artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscalable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that these types of novel enzymes are a possible product.

[Co(dmg)2Cl2] binding control study with α3W S10 Table S1 Summary of protein and cobalt concentrations S2

Table S4
Time intervals for determination of initial rates S7 Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2024

I. α3C protein sequence
The cysteine that is functionalized with methylpyridine is shown underscored in bold text.

II. Determination of protein concentration
The protein concentration was determined either by the Bradford assay, using the Bio-Rad Protein Assay kit II (Bio-Rad Laboratories inc.), or with the Pierce TM BCA Protein Assay Kit (Thermo Scientific).The Bio-Rad Protein Assay was not compatible with cobaloxime functionalized protein, 3, so the BCA assay was used to determine the concentrations of protein in the product.As a standard protein solution, we used purified α3W instead of the provided bovine serum albumin.The α3W protein concentration was determined from the optical density at 280 nm and molar absorption coefficient of 5500 M -1 cm -1 .The BCA enhanced test-tube procedure was performed as described, except that all volumes were halved in comparison to the provided instructions.By comparing the protein quantification and the cobalt quantification (vide infra), it was found that the cobalt:protein ratio is ranged from 1:1.20-1:2.68.More specifically for the three batches of protein indicated here, the average cobalt:protein ratio was 1:1.79 (Table S2).The colorimetric Pierce TM BCA Protein Assay is less sensitive than ICP-OES which is used to determine the cobalt concentration.

III. Determination of cobalt concentration by ICP-OES.
The cobalt content in samples containing 2 and 3 was quantified using inductively coupled plasma -optical emission spectrometry (ICP-OES).This method was used for all samples of 2 and 3 subject to chemical reduction by [Eu(EGTA)] 2-; the cobalt concentration in samples used for photocatalysis and electrochemistry were subject to the assay described in section IV below.After the termination of each hydrogen evolution experiment the solution was sampled by ICP-OES.The concentration and relative standard deviation (RSD) for each sample is given in Table S1 with an identification number.These identification numbers are correlated to the numerical labels shown in Figure S6.
The ICP-OES experiment was performed with the Avio 200 ICP-optical emission spectrometer, (Perkin Elmer, Inc.).The presence of Co was confirmed by the emission at 228.616 nm where the peak was defined by three points.The samples for ICP-OES analysis were prepared by mixing equal volumes of the sample solution and 4 wt% concentrated nitric acid in milliQ water.The diluted samples were filtered with a 0.2 µm syringe filter.The sample cobalt concentrations were quantified against a calibration curve prepared from a commercial standard solution CPAchem of cobalt (100 mg/L Co in 2% HNO3 matrix).Standards of lower concentration were prepared by appropriate dilution.The resulted calibration line described with 0.999918 correlation coefficient.All measured concentrations used to report quantities have RSD <3%.S1).Circles are averaged data obtained from three replicate spectra.Grey lines are non-linear curve fits used to determine the stability of the protein in the absence of denaturant.Table S3.Voltametric peak potentials for 2 and 3, from the cyclic voltammograms in figure 2D.

V. Calculation of free Eu(II) in solutions of Eu(EGTA)
Ethylene glycol tetraacetic acid, EGTA, is a tetradentate ligand with four groups subject to protonation.The pKa values associated with these groups are pK1-pK4 = 2.0, 2.68, 8.85, 9.43. 4 The concentration of EGTA 4-, the form which binds Eu(II), depends on the buffer pH.The concentration of EGTA 4-, can be determined using equation 14 from reference 3: where α H is the partition coefficient at a given hydrogen ion concentration, EGTA´ is the total amount of EGTA not bound to metal and (EGTA) is the total concentration of dissociated anion.The log of the stability constant,  !"!#$% !"('') , was reported from electrochemistry to be 9.38. 5Equation 15 from reference 3 is used to determines the apparent stability constant as a function of pH: where (EuEGTA) is the concentration of [Eu(EGTA)] 2-in solution.Then using equation 6 from reference 3 the fraction of free metal in the solution can be calculated.
where (ETGA)t and (Eu)t are the total concentration added to the solution.Finally one can calculate the concentration of [Eu(EGTA)] 2-from (Eu)t=(Eu)+[Eu(EGTA)].

VI. Determination of initial rates from hydrogen evolution measurements
Table S4.Time intervals for determination of initial rates, under given experimental condition.Figure 2B showing H2 produced by 2 (a, orange) and 3 (b, teal) at pH 6.2 under photocatalytic conditions.Detection by H2 sensing electrode.The shaded area shows which data were used for linear fits to determined rates of hydrogen production reported in Table 1.S1.The shaded grey areas show which data were used for linear fits to determined rates of hydrogen production reported in Table 1.

IX. Binding control -[Co(dmg)2Cl2] and α3W
We performed a binding control experiment using α3W and [Co(dmg)2Cl2], 1, to test for potential cobaloxime binding to the protein scaffold that may occur at sites other than site 32.α3W was treated with 1 under conditions identical to those used to bind 1 to 3-MePy-α3C.Briefly, α3W was dissolved in pH 8.5 buffer containing 2 M guanidinium.A 10-fold molar excess of [Co(dmg)2Cl2] and 45-fold excess of reducing agent (TEA) were added to the protein solution and incubated under argon atmosphere for 4 hours at RT.The cobalt concentration in these samples was 0.87 mg/L.The reaction products were subsequently dialyzed against water in 3.5 kDA dialysis tubing.ICP analysis, with resolution of 0.0001 mg/L, was carried out on dialyzed protein samples.No cobalt was detected.From this experimental control we can conclude that under our experimental conditions, [Co(dmg)2Cl2] does not bind to the noncoordinating amino acids in the α3 scaffold.

Figure S3 .
Figure S3.Chemical denaturation of α3W, α3C and 3 in 50 mM KPi pH7.Folding/unfolding transition induced by addition of urea to: A) a sample of 19.1 µM α3W, B) a sample of 18.8 µM α3C, and C) a sample of 30.6 µM 3 (from Sample C, TableS1).Circles are averaged data obtained from three replicate spectra.Grey lines are non-linear curve fits used to determine the stability of the protein in the absence of denaturant.3

3
Figure S3.Chemical denaturation of α3W, α3C and 3 in 50 mM KPi pH7.Folding/unfolding transition induced by addition of urea to: A) a sample of 19.1 µM α3W, B) a sample of 18.8 µM α3C, and C) a sample of 30.6 µM 3 (from Sample C, TableS1).Circles are averaged data obtained from three replicate spectra.Grey lines are non-linear curve fits used to determine the stability of the protein in the absence of denaturant.3

Figure S5 .
Figure S5.Photochemical hydrogen evolution detected by Clark-type hydrogen sensor.Figure2Bshowing H2 produced by 2 (a, orange) and 3 (b, teal) at pH 6.2 under photocatalytic conditions.Detection by H2 sensing electrode.The shaded area shows which data were used for linear fits to determined rates of hydrogen production reported in Table1.

Figure S6 .
Figure S6.Chemical hydrogen evolution detected by Clark-type hydrogen sensor.Hydrogen evolved by 2 and 3 in the presence of chemical reducing agent [Eu(EGTA)] 2-.Light traces are individual measurements; dark traces are the average of repeated measurements.Traces 1-15 were recorded on the same day and therefore more directly comparable.Traces 16-18 were recorded on a different day.Numbers correspond to the sample number of TableS1.The shaded grey areas show which data were used for linear fits to determined rates of hydrogen production reported in Table1.A. Complex 2, pH 7. B. Complex 2, pH 8. C. Complex 3, pH 7. D. Complex 3, pH 8. E. Complex 3, pH 7 on different day.F. All hydrogen evolution traces (4-7 -teal, traces 4-7) and (16-18 -lilac, traces 16-18) for 3 at pH 7. The trace shown in pink (star) is the average of all purple and teal traces in the plot.

A. Complex 2 ,
Figure S6.Chemical hydrogen evolution detected by Clark-type hydrogen sensor.Hydrogen evolved by 2 and 3 in the presence of chemical reducing agent [Eu(EGTA)] 2-.Light traces are individual measurements; dark traces are the average of repeated measurements.Traces 1-15 were recorded on the same day and therefore more directly comparable.Traces 16-18 were recorded on a different day.Numbers correspond to the sample number of TableS1.The shaded grey areas show which data were used for linear fits to determined rates of hydrogen production reported in Table1.A. Complex 2, pH 7. B. Complex 2, pH 8. C. Complex 3, pH 7. D. Complex 3, pH 8. E. Complex 3, pH 7 on different day.F. All hydrogen evolution traces (4-7 -teal, traces 4-7) and (16-18 -lilac, traces 16-18) for 3 at pH 7. The trace shown in pink (star) is the average of all purple and teal traces in the plot.

Figure S8 .
Figure S8.PAR assay for cobalt quantification.A. Spectra of PAR with increasing concentrations (0-20 µM) of CoCl2 B. Difference spectra of [Co(PAR)n] 2+ (n =1, 2) calculated by subtracting the spectrum in of PAR with no added cobalt from all other spectra.C. Spectra of PAR with increasing concentrations (0-20 µM) of [Co(dmg)2Cl2].D. Difference spectra of [Co(PAR)n] 2+ calculated by subtracting the spectrum of PAR with no added cobalt from all other spectra.E. Absorbance at 412 (circles) and 510 (triangles) nm as a function of CoCl2 (green) and [Co(dmg)2Cl2] (pink) concentration.

Table S1 .
Comparison of cobalt and protein concentration for three independently prepared batches of 3.

Table S2 .
In situ cobalt concentration in each sample used for hydrogen evolution experiments, quantified by ICP-OES. Figure S6 shows sample number for each individual trace.Complex 2 = [Co(dmg)2(py)Cl] and complex 3 = [Co(dmg)2(3-MePy-α3C)Cl].Entries for [Co] with multiple values correspond to repeat measurements with average given in parentheses.