Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.

(S1) 345  (S4) These calculations were performed for the initial spectra of the GSH titration (with the Cu(II)GHK spectrum subtracted for the reasons stated above). A satisfactory spread of the values was obtained, except of 10 mM GSH, giving an average value of 0.082, as shown in Table S1. The ∆ 300 value was also time-corrected with regard to the time point of the A345 measurement using the equation obtained to extrapolate the data for the reduction process, as described below.
(2) Extrapolation to zero time point ( 300 0 and 345 0 ) 300 0 and 345 0 values were acquired by individual extrapolations at given wavelengths using the appropriate 1 st order kinetic equation fitted to four initial reaction time points, separately for each GSH concentration (Table S5). The R 2 was 0.9992 ± 0.0017 for A300 and 0.9974 ± 0.0080 for A345.
The apparent paradox of zero-time presence of Cu(I)4GSH6 is justified by a very fast initial rate of Cu(II) reduction; see main text for details of reaction course and mechanism. (

3) Determination of [Cu(I)4GSH6] and [GSH-Cu(II)GHK] at 0 s and stability constant for the GSH-Cu(II)GHK complex
Based on the data obtained in previous steps, the concentrations of both complexes were calculated for each GSH concentration separately according to the following set of equations: results of which are listed in Table S5.
The calculated values were applied to obtain the GSH-Cu(II)GHK ternary stability constant (S11) from the reaction (S10): [GSH]·[Cu II GHK] (S11) In these calculations, the minor contribution of GHK-Cu(II)GHK was ignored, as speciation calculations indicated that its contribution never exceeded 1.4% of total copper in the reaction. The residual GSH concentration was calculated assuming 1 GSH molecule being consumed per one GSH-Cu(II)GHK complex and 1.5 GSH equivalent being engaged into one Cu(I) ion coordination (in accord to Cu(I)4GSH6). Cu(II)GHK concentration was determined as a difference between total Cu (0.45 mM) and calculated concentrations of GSH-Cu(II)GHK and Cu(I) (Table S5).

EPR
The EPR spectra were acquired at 77K using a CMS8400 X-band (9.42 GHz) spectrometer (Adani, Belarus) fitted with a TE102 cavity and a quartz cold finger insert (Wilmad, WG-816-B-Q). Measurements were made using the following settings: microwave frequency, 9.42 GHz; microwave attenuation, 10 dB; magnetic field sweep rate, 8 gauss s -1 ; magnetic field modulation amplitude, 8 gauss; magnetic field modulation frequency, 100 kHz; field resolution, 4096 points; receiver gain, 100; time constant, 100 ms; averages, 10. Baseline correction of spectra was carried out by subtracting the spectrum of 10% glycerol in water. Normalization of the mic rowave absorption to unit area was carried out by double-integration of the experimental first-harmonic spectra.

NMR
The NMR experiments were performed at 298 K utilizing a Varian Inova 500 NMR spectrometer operating at 11.7 T magnetic field ( 1 H resonance frequency 500.606 MHz) equipped with three channels, z-gradient unit Performa IV, and 1 H/ 13 C/ 15 N triple resonance probehead with inverse detection. The NMR sample contained initially 2 mM GSH and 0.5 mM GHK dissolved in 90%/10% H2O/D2O, 50 mM sodium phosphate buffer, at pH 7.4 to which 0.45 mM Cu(NO3)2 was added, followed by a pH check. The control sample, without GHK, was prepared under the same conditions. Additional control samples contained pure 1 mM GHK, GSH and GSSG dissolved in the same buffer. The samples were measured in a side-by-side regime. Assignments of the 1 H and 13 C resonances were based on a joint analysis of one-dimensional 1 H spectra, two-dimensional 1 H-1 H TOCSY (80 ms mixing time), and 1 H-13 C HSQC recorded on the natural abundance of the 13 C isotope. All spectra were referenced indirectly with respect to external sodium 2,2dimethyl-2-silapentane-5-sulfonate (DSS) using Ξ = 0.251449530 ratio for the 13 C dimension. 2 Translation diffusion coefficients (Dtr) were measured with Pulsed Field Gradient Spin Echo NMR (PGSE NMR) experiment at 298 K. The values of Dtr were independently obtained for two signals at 3 and 3.15 ppm. The raw PGSE data was initially manipulated using MestReNova software to correct phase shifts as well as base-line using the Bernstein Polynomial Fit method. Next, extracted spin echo signal decays at 3 and 3.15 ppm were linearized vs G 2 and diffusion coefficients were obtained from the slope of the linear fit using Origin 2021 software. The data points were fit to the general Stejskal-Tanner equation (S12): 3 where γ is the 1 H gyromagnetic ratio, Δ is diffusion time [s], G is the magnetic field gradient strength [T/m], and δ is gradient duration [s]. The experimental data were acquired in 25 gradients steps, using diffusion time 50 ms, and gradient duration was set typically to 2 ms. The DOSY spectra and Dtr values were also calculated using the Bayesian method 4 embedded in MestReNova software 12.04.

Electrochemistry
Electrochemical measurements were performed using the CHI 1030 potentiostat (CH Instrument, Austin, USA) in a three-electrode arrangement: Ag/AgCl electrode as a reference electrode, platinum wire as an auxiliary electrode, and glassy carbon electrode (GCE, BASi, 3 mm diameter) as a working electrode. Prior to each measurement, the GCE surface was carefully polished to a mirror-like surface with the use of alumina suspensions (1.0 and 0.3 µm in succession) on a Buehler polishing cloth. To remove residual abrasive particles, the working electrode was sonicated for 1 min and then rinsed thoroughly with distilled water. The conductivity of the solution was provided by 100 mM KNO3, therefore strict control of pH was demanded (small aliquots of concentrated KOH or HNO3 solutions were used, if required). To avoid the undesired influence of dissolved oxygen and free copper ions, all electrochemical exper iments were carried out under argon with an excess of the ligand (peptide-to-copper(II) ratio was 1.0:0.9). Cyclic voltammetry (CV) curves at different scan rates and different potential ranges were recorded.  Table S2. Spin Hamiltonian parameters used to simulate the EPR spectra in Figure S18 and comparison with previous studies at room temperature.      represents changes that occurred within the mixing time (expressed as a difference between the first spectrum recorded after GSH introduction and Cu(II)GHK spectrum), the black line represents overall changes that took place during the reaction (expressed as a difference between the last spectrum recorded after GSH introduction and Cu(II)GHK spectrum), the red line represents changes that took place during Cu(II)GHK complex recovery step (expressed as a difference between the spectrum recorded after 40 min and immediately after GSH introduction); pH=7.4, T=37°C.               Table S2.  Figure S20); pH=7.4. After 60 minutes of storage on ice, the sample was incubated for yet 2 hours at 37°C (to make sure the reaction is completed), after which the last EPR spectrum was recorded. The total concentration of Cu(II) species was determined by double integration of the first harmonic spectra.       Comments: The final spectra of both samples are identical, except for the acetate signal derived from the GHK formulation, and are dominated by the signals of GSSG. This means that in the presence of GHK, all Cu(II) initially reduced by GSH is reoxidized and bound to GHK, causing broadening out of its signals. Only trace signals of Lys protons of GHK can be noticed at high field. Figure S28. Comparison of DOSY spectra recorded for the selected spectra presented in Figure S24, at the start, 22.5 and 93 h. Lines mark signals exhibiting the same diffusion coefficient.