Pre-Steady State Kinetics of Catalytic Intermediates of an [FeFe]-Hydrogenase

Transcription

1 Supporting Information Pre-Steady State Kinetics of Catalytic Intermediates of an [FeFe]-Hydrogenase Brandon L. Greene, Gerrit J. Schut, Michael W. W. Adams and R. Brian Dyer * Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA *Corresponding author: briandyer@emory.edu

2 Table of Contents Materials and Methods. Supplemental Discussion. FTIR peak assignment. Table S1. CO and CN peak comparison for various [FeFe] H 2 ases. Figure S1. Schematic representation of the [FeFe] H 2 ase of Thermotoga maritima Figure S2. FTIR spectra of Tm H 2 ase under 100% H 2 and 100 % N 2. Figure S3. FTIR spectra of Tm H 2 ase under 100% N 2 and 100% CO. Figure S4. FTIR of steady state photolysis of the Tm H ox state. Figure S5. TRIR of photolysis of H inact, H red and H sred. Figure S6. TRIR of photolysis of H ox and H ox -CO. Figure S7. Initial FTIR spectrum of photo-reduction sample. Figure S8. Time dependent FTIR spectra of auto-oxidation. Figure S9. UV-Vis spectra of Tm H 2 ase sample and Mb reference before and after photolysis. Figure S10. Visible MV + transients for Tm H 2 ase sample and Mb reference. Table S2. Visible transient fit parameters. Figure S11. IR H red transients and associated fits.

3 Materials and Methods. Thermotoga maritima [FeFe] H 2 ase expression and purification. The Tm [FeFe]-H 2 ase was purified as described in Ref. 1. Sample preparation for spectroscopy. Steady state measurements: Samples for steady state spectroscopy were prepared anaerobically in a 5% H 2 in N 2 atmosphere in a glove box (Coy Laboratory Products). Tm H 2 ase stock samples were buffer exchanged into 100 mm KP i ph = 7.0 containing 10 mm MV 2+ (Sigma Aldrich) by eight 1:5 concentration dilution steps in an amicon 50 kda molecular weight cutoff filter (EMD Millipore), which immediately turned blue due to reduction by the enzyme. For H 2 reduced samples 10 mm NADH (Sigma Aldrich) was also added to the buffer. Approximately 2-3 mg of protein was concentrated to 20 µl by spin concentration and transferred to a septum sealed glass vials for gas exchange. Samples for N 2 and CO atmospheres were initially gas exchanged with 100% N 2 for 2 minutes by passive air flow over the headspace then allowed to equilibrate for 10 minutes which caused the blue color to slowly dissipate. For the N 2 sample a second 2 minute passive flow of N 2 was performed to further remove H 2, whereas CO samples were exposed to a passive stream of 100% CO for 2 minutes. For H 2 reduced samples, a similar procedure was performed, but passive flow of 100% H 2 was used rather than N 2 and only a single 2 minute exposure was used. Following gas exchange and equilibration for >10 minutes, the samples were then transferred back into the anaerobic chamber for loading of the FTIR cells, with care taken to not expose the samples during transfer to the 5% H2 in N2 atmosphere by quick transfer via gas tight syringe. A 50 µm path length spacer separating two CaF 2 windows was used for all samples and the buffer served as a reference for computing the protein absorbance. Transient measurements: Samples were prepared as previously reported with minor modifications. 2 All sample preparation and handling was performed at room temperature under a 4% H 2 in N 2 atmosphere as above. Stock solutions of Tm H 2 ase were buffer exchanged into 100 mm KP i buffer ph 7.0. Once DTT from storage buffer was removed, the H 2 was flushed from the anaerobic chamber by pure N 2 to below 1%. Next, the Tm H 2 ase sample was buffer exchanged into the same buffer, supplemented with 10 mm MV 2+ and 12 mm NADPH by 4 further concentration dilution steps. In the H 2 free environment, no MV 2+ reduction was observed. Finally, the Tm H 2 ase was concentrated to ~15 µl to a final concentration of ~1.5 (3 mg) mm and loaded via a gas tight syringe into half of an air tight IR cell consisting of two CaF 2 windows mounted in a copper holder and a 75 µm spacer. Additionally, horse heart myoglobin (metmb, Sigma Aldrigh) was dissolved in the anaerobic MV 2+ and NADPH containing buffer and similarly concentrated to ~50 µl of 1 mm metmb and loaded into the other half of the IR cell. Fourier transform infrared spectroscopy (FTIR). FTIR spectra were collected as previously reported with minor modification. 2-4 Briefly, the anaerobically prepared Tm H 2 ase sample in the IR cell was mounted onto a temperature controlled copper block in the external compartment of a Varian Excalibur FTIR spectrometer equipped with a liquid nitrogen cooled MCT detector (Kolmar Technologies, Inc.). Both sample and reference sides of the split IR cell were measured for background correction and the sample was temperature controlled by a recirculating water bath and the temperature was measured directly on the cell. Spectra were collected at 2 cm -1 resolution averaging 2048 scans unless otherwise stated, and baselines were subtracted by spline

4 functions. Care was taken to avoid weighting spline baselines to CO/CN peak rich regions to avoid spectral distortion. To limit the time between steady state and transient measurements, only 128 scans were averaged for the first spectrum prior to transient measurements (Fig. S7), resulting in lower signal to noise ratio relative to subsequent FTIR measurements. Ultraviolet-visible (UV-Vis) spectroscopy. UV-Vis measurements were performed identically as previously reported. 2 Time resolved infrared/visible spectroscopy. The simultaneous nanosecond time resolved infrared/visible instrument is described in detail elsewhere. 2 Kinetics were measured at room temperature and the reported transients represent the average of 50 single shots on independent sample points, achieved by rastering the sample between single shots. After complete exposure, the sample and reference were then re-exposed resulting in the 2 nd shot data and so on. The 355 nm pump was set to a power density of 300 mj/cm 2. The IR solvent heating signature resulting from non-radiative excited state decay of the NADPH/Tm H 2 ase from the sample was subtracted by transients collected on the reference cell at the same wavelength as previously reported. 2-4 For photolysis measurements, between shots were averaged at each wavelength at individual spots on the sample and the data was analyzed similarly as above using an appropriate reference solvent heating signature (typically transients collected at 1910 cm -1 ).

5 Supplemental Discussion. FTIR peak assignment in the as prepared Tm [FeFe] H 2 ase sample reported in the main text Fig. 1. The terminal CO (ν CO ~ cm -1 ) region of the spectrum is typically the easiest to identify due to the strong dipole moment of the transition and significant π-back bonding which modulates the observed vibrational frequency in response to redox changes of the active site. In contrast, the terminal CN ligands (ν CO ~ cm -1 ) suffer from lower oscillator strength as opposed to CO and less π-back bonding. Two vibrational resonances in the terminal ν CO region can be associated with a single enzyme redox state, assuming an active site configuration of (CO t ) 2 (CN t ) 2 (CO b ), where t indicates terminal and b indicates bridging. It is known that the bridging CO ligand can become terminally coordinating to the distal iron upon active site reduction, increasing the number of terminal CO ligands, but this resonance is observed <1900 cm -1. To characterize the steady state intermediates of the Tm [FeFe] H 2 ase, we prepared enzyme samples under 100 % H 2, 100% N 2 and 100% CO atmospheres and examined them by FTIR. The H 2 and N 2 incubated samples are displayed in Figure 1 of the main text and S2 and the CO and N 2 incubated samples are compared below in Figure S3. Under a N 2 atmosphere, 8 terminal CO resonances are clearly resolved with some shoulder features which are not well resolved. The most intense absorption features are located at 2007 cm -1 and 1954 cm -1, with smaller features at 2023, 1999, 1980, 1971, 1942 and 1931 cm -1 as well as shoulders to the 1954 cm -1 band on both high and low energy sides. Furthermore, there are four CN peaks at 2098, 2092, 2080 and 2074 cm -1 and one clear bridging CO resonance at 1824 cm -1. The peak at 2006 was observed to be highly correlated with the broad band at 1980 cm -1 in numerous samples from various enzyme preps and conditions, and was thus assigned to an inactive form the H- cluster termed H inact. Indeed, this species has been observed previously in the Dv (1848, 1983, 2008, 2087 and 2106 cm -1 ) 5 and Dd (1848, 1983, 2007, 2084 and 2106 cm -1 ) enzymes. 5-8 This state is proposed to results from oxidative damage of the H-cluster either by air or by anodic electrochemical potentials. It is also possible that the broad resonance at 1980 contains contributions from the intermediate state H trans (1836, 1977, 1983, 2075 and 2100 cm -1 for Dd), another more oxidized species than H ox. 5-8 To the red of the 1980 and 2006 cm -1 resonances, the small peak at 1971 and shoulder at 1964 cm -1 represents H ox -CO (see Figure S3, S4) which also has contributions at 1810 and 2009 cm -1 which compare favorably to other [FeFe] H 2 ases (1810, 1963, 1971, 2016, 2088, 2094 cm -1 for Dd; 1810, 1964, 1972, 2084 and 2092 cm -1 for Cr and 1810, 1971, 1974, 2017, 2077 and 2095 cm -1 for Cp). The peak at 1965 cm -1 has been assigned in other enzymes to the most oxidized steady state that is proposed to be active in catalysis, namely H ox. In Dd, the proton-coupled reduction of H ox, forming H red also yields a resonance at 1965 cm -1, thus the peak at 1965 cm -1 must be examined within the broader context of the overall active site FTIR spectrum. The resonance at 1940 cm -1 is observed to bleach upon extended 351 exposure concomitant with formation of H ox - CO. This phenomenon has been observed in Dd and is associated with H ox cannibalism in the presence of light, a phenomenon unique to this state. 9 In these photolysis measurements, bleach signatures are also observed at 1800, 2080 and 2075 cm -1 consistent with the bridging CO and terminal CN groups respectively. The Tm H2ase samples prepared under H2 reveal some spectral changes as expected for significant enzyme reduction (see Figure 1 main text and S2). In these samples it should be possible to identify the further reduced states including H red and possibly H sred. New features at

6 2036, 1921 and 1888 cm -1 are observed in these samples suggesting that they belong to one of the aforementioned states, but definitively assigning these resonances is challenging based on this data alone. In Cr, H red gives rise to ν COt resonances at 1935 and 1891 cm -1, both of which are observed in the spectrum (1931 and 1888 cm -1 ) as well as a weak feature at 1791 cm -1, similar to the ν COb resonance in Cr of 1793 cm -1. These data are consistent with an Hred assignment similar to Cr. Additionally, in Cr, the H sred state has been observed to have a CO t resonance at 1954, 1919 and 1882 cm The spectrum of Tm [FeFe] H 2 ase also displays peaks at 1954, 1922 and 1888 cm -1 which would support an H sred assignment. Interestingly, the relative intensity of the Hsred features appears stronger than that of H red, and features of H ox -CO and H ox have nearly vanished, consistent with this hypothesis. Based on both the spectral similarities between the Tm H2ase and other enzymes as well as the observed kinetics we propose the state assignment detailed in Table S1. In summary, the ν COt and ν COb/t regions of the spectrum are fully consistent with a mixture of H ox, H red and H sred which are, spectrally, nearly identical to those observed in Cr and similar to Dd, as well as inactive species such as H inact, H ox -CO and possibly H trans. H inact and H trans have not been explored to our knowledge in Cr and therefore their comparison at present cannot be provided. The above assignments are further supported by examination of the FTIR spectrum upon auto-oxidation resulting from incubation in the IR cell. As shown in Fig. S5, the bands associated with H ox, H inact and H trans as well as a new species termed H inact are observed to increase over time, whereas peaks associated with H red and H sred disappear. Based on the general similarity of the overall FTIR spectrum to other known [FeFe] H 2 ases, we expect their active site electronic and stereo-chemical configurations are similar. Table S1. CO and CN peak summary of various characterized [FeFe] H 2 ases. Redox State Organism ν CNt1 (cm -1 ) ν CNt2 (cm -1 ) ν COt1 (cm -1 ) ν COt2 (cm -1 ) ν COb/t (cm -1 ) H inact Tm * 1845 Dd Dv H trans Tm 1 n.o. n.o. 1980* 1980* 1830 Dd H ox Tm Dd Cr H red Tm 1 n.o. n.o * 1793 Dd Cr H sred Tm * Dd 2 n.o. n.o Cr * Indicate broad peaks for which the peak absorbance likely represents multiple components. 1 this work, 2 ref. 7, 3 ref. 5, 4 ref 11,12. n.o. indicates not observed.

7 Figure S1. Schematic representation of the Thermotoga maritima [FeFe]-H 2 ase. Proposed electron transfer pathways are shown in arrows, although other electron transfer pathways are possible. Cofactors; FMN, flavin mononucleotide; FexSx, iron sulfur clusters; H-cluster, active site cofactor; NADH, nicotinamide adenine dinucleotide and Fd, ferredoxin.

8 Figure S2. Peak assignments of H ox, H red/sred, H inact and H ox -CO based on FTIR measurements under 100% H 2 (blue) and 100% N 2 (red) conditions (data identical to figure 1 of main text) as well as the 100% CO spectrum (Fig. S3), steady state photolysis data (Fig. S4), photo-reduction data (main text Fig. 3 and S7) and similarity with other [FeFe] H 2 ases (see Discussion above).

9 Figure S3. FTIR spectra of Tm [FeFe] H 2 ase in the presence of 100% N 2 and 100% CO and assignment of H ox -CO. Peaks associated with the binding of CO are indicated by dashed lines. The H ox state absorbance at 1940 cm -1 decreased concomitantly. Of note is the slight shift of the 2007 band to higher frequency, consistent with subsequent measurements of the slightly higher frequency (2009 cm -1 ) resonance associated with the exogenous terminally bound CO of H ox - CO.

10 Figure S4. FTIR characterization of steady state photolysis of Tm H 2 ase incubated under 100% CO. Original dark spectrum is shown in black and difference spectrum (pre illumination post illumination, multiplied by 5) after 2 minutes of illumination (2.5 mw of 351 nm light generated by an Nd:YLF operating at 2.5 khz) shown in gray, with dashed lines indicating correlation between pre and post photolysis features. Total power delivered was ~3 J/cm 2. Note that the H ox - CO peak at 2009 cm -1 is shifted from the H inact peak slightly at 2007 cm -1, which also has a broad band at 1980 cm -1.

11 Figure S5. TRIR photolysis dynamics of Tm H 2 ase under 100% CO (see Figure S3) of H inact (blue, 1978 cm -1 ), H sred (green, 1954 cm -1 ) and H red (red, 1930 cm -1 ) following photolysis by the 355 nm pump pulse of the Nd:YAG laser. Excitation power was 300 mj/cm 2 and 500 single shots were averaged per spot. Background solvent heating signature was subtracted from transients collected at 1910 cm -1 and was within 10% of the amplitude of the signal requiring little normalization.

12 Figure S6. TRIR transients Tm H 2 ase prepared under 100% CO (see Figure S3) of H ox (left, 1940 cm -1 ) and H ox -CO (right, 1965 cm -1 ) following photolysis by the 355 nm pump pulse of the Nd:YAG laser. Excitation power was 1.5 J/cm 2 and 500 single shots were averaged at five unique sample locations (100 shots per spot). Background solvent heating signature was subtracted from transients collected at 1910 cm -1 and was within 10% of the amplitude of the signal requiring little normalization.

13 Figure S7. FTIR spectrum of Tm H 2 ase sample used for photoreduction measurements reported in the main text. Sample (~1.5 mm) prepared under N 2 in the presence of 10 mm MV and 12 mm NADPH in 100 mm KP i ph = 7.0. FTIR spectrum collected at 2 cm -1 resolution averaging 128 scans in an 80 µm path length cell and referenced to 1 mm metmb in the same buffer. Major peaks aside from H inact (2006 and 1980 cm -1 ) and H ox -CO (2008, 1973, 1964 cm -1 ) include H ox (1965 and 1940 cm -1 ), H red (1930 and possibly 1888 cm -1 ) and H sred (1954, 1921 and 1888 cm -1 ).

14 Figure S8. Time dependent FTIR analysis of the Tm [FeFe] H 2 ase sample shown in Fig. S4 after 14 days of incubation at RT. (A) Time dependent FTIR spectra recorded as prepared (dark red, top), after 1 day (next down), 3 days, 5 days, 7 days, 9 days and 14 days (dark blue, bottom). Vertical lines were added as visual aid for species present at any time during data collection. (B) Difference FTIR spectrum of the sample after 14 days vs. after 3 days. Negative peaks correspond to redox states which decayed after day 3 and positive bands correspond to new features after 14 days. The sample showed no evidence of aggregation optically, and the integrated ν CO intensity was nearly constant over the course of the data collection. This is as expected since the enzyme comes from a hyperthermophile, making it highly stable at RT. H ox - CO was not observed to form to any significant extent following kinetics measurements (top two spectra, panel A), suggesting minimal photolysis of the endogenous CO ligands of the active site.

15 Figure S9. UV-Vis absorbance of the Tm H 2 ase sample (red) and metmb reference (blue) before (solid lines) and after (dashed lines) photo-reduction experiments.

16 Figure S10. Transient visible kinetics probed at 635 nm (MV + ) for both Tm H 2 ase and myoglobin. (A) Transient dynamics of Tm [FeFe] H 2 ase after 1 (red), 2 (orange), 3 (green), 4 (blue) and 5 (purple) laser excitation pulses and associated bi-exponential fits. Offsets of 5 mod added for clarity. (B) Transient dynamics of myoglobin from 1 (red), 2 (orange), 3 (green), 4 (blue) and 5 (purple) laser excitation pulses and associated bi-exponential fits. Offsets of 10 mod added for clarity.

17 Table S nm kinetics as a function of IR probe wavelength and transient flash number. A x indicates the amplitude of fit component x and k x indicates exponential decay rate for fit component x. wavenumber k 2 (s -1 ) offset (mod) Laser pulse A 1 k 1 (s -1 ) A 2 (cm -1 ) (mod) (mod) Sample , Sample , Sample , Sample Sample , Reference , Reference , Reference , Reference , Reference ,

18 Figure S11. Transient IR trace for H red (1931 cm -1, red) with associated mono- (black dashed line) and bi-exponential (black solid line) fits. Variance for mono- and bi-exponential fits were 2.75 and 2.71 x 10-5 respectively, which are statistically identical.

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