To determine the charge transfer efficiency of DET, methyl viologen (MV) as an electron mediator is used to activate MET (Figure ). Upon the addition of MV (2 mM), the H2 yield reaches 24.0 ± 0.1 µmol for CpI [FeFe]-H2ase and 25.6 ± 1.3 µmol for DvH [NiFeSe]-H2ase after 24 h of irradiation. The comparable H2 yields despite differences in specific activity indicate that the rate limiting step during MET is not enzyme turnover but the photoactivity of NCN CNX, as evidenced by control experiments (Table ). The efficiency of DET is qualitatively determined by the DET:MET ratio. In the case of [FeFe]-H2ase and [NiFeSe]-H2ase, DET:MET ratios of approximately 25% and 0.5% are observed, respectively (Figure ). By comparing the results obtained from DET and MET, a schematic representation can be depicted in Figure . It highlights the establishment of efficient electron transfer directly between NCN CNX and [FeFe]-H2ase, with a DET:MET ratio of 25%. This finding emphasizes the effectiveness and benefits of electrostatic interactions in facilitating DET. Conversely, the electrostatic repulsion between [NiFeSe]-H2ase and NCN CNX prevents DET, resulting in a low DET:MET ratio of 0.5%. To gain deep insights into the interaction between NCN CNX and H2ase, QCM analysis was conducted. As shown in the schematic in Figure , the Au-coated quartz chip was functionalized with a thin layer of NCN CNX by drop casting 0.5 mL of an ultrasonicated suspension (0.1 mg mL -1 ) of NCN CNX, to mimic the operando conditions during photocatalysis. By flowing a buffer solution containing enzymes on the chip, the adsorption process of H2ase at the surface of NCN CNX can be monitored as a function of time and quantified based on the Sauerbrey equation. Figure shows the QCM analysis of CpI [FeFe]-H2ase and DvH [NiFeSe]-H2ase on the NCN CNX-modified chip. After establishing a stable baseline by circulating 0.1 M MOPS pH 7 buffer with 50 mM 4-MBA, the enzymes were introduced separately at the same concentration as in the photocatalysis experiments. The adsorption of both H2ases on NCN CNX exhibits in two distinct stages, a fast adsorption process before 1.5 h and a slow adsorption process after 1.5 h. Interestingly, a higher amount of [NiFeSe]-H2ase (39.5 pmol cm -2 ) is adsorbed onto NCN CNX compared to [FeFe]-H2ase (16.6 pmol cm -2 ) over 10 h. The observed adsorption profiles can be explained by the larger dipole moment of [NiFeSe]-H2ase (1972 D) compared to [FeFe]-H2ase (1707 D), with the orientation of the dipole moment explaining the improved electroactive orientation of [FeFe]-H2ase on negatively charged surfaces (Figure ). For [NiFeSe]-H2ase, DET can only be established via the negatively charged patch near the distal FeS cluster, which is unfavorable for the negatively charged NCN CNX and thus dramatically reduces DET to the enzyme active site for catalysis. Consequently, the strong association observed at the NCN CNX|[NiFeSe]-H2ase interface is non-specific and results mainly in inactive biohybrid assemblies. Therefore, QCM analysis provides valuable insights into the significance of specific interactions in facilitating DET. To investigate the charge carrier dynamics between H2ases and NCN CNX, PEIS was performed using a three-electrode configuration. By applying a sinusoidal potential modulation to the NCN CNX-modified working electrode, which was made by depositing a NCN CNX suspension (5 µL, 24 mg mL -1 ) on FTOcoated glass, the impedance was recorded as the ratio of the complex-valued potential and current. A Randles circuit consisting of a series resistance (𝑅 ! ) in parallel with a combination of bulk capacitance (𝐶 "#$% ) and charge transfer resistance (𝑅 &' ), was proposed to fit the impedance response (Figure ). The Nyquist plot (Figure ) of the impedance response measured at -0.1 V vs the reversible hydrogen electrode (RHE), is dominated by a single semicircle with no indication of a Warburg diffusion element. The proposed equivalent circuit provided a good fit (r 2 > 0.95) to the impedance response, enabling quantitative analysis of the charge transfer process. Upon introduction of [FeFe]-H2ase on the working electrode, a decrease in the semicircle diameter is observed, corresponding to a decrease in 𝑅 &' from 15,975 Ω to 12,317 Ω. This indicates that [FeFe]-H2ase, as a biocatalyst with the correct orientation, facilitates the charge transfer from NCN CNX to the electrolyte for HER. Likewise, the non-specific interaction between [NiFeSe]-H2ase and NCN CNX results in a 𝑅 &' of 13,950 Ω, similar to the bare NCN CNX. Such behaviors have been widely observed when incorporating synthetic co-catalysts onto semiconductors. The fitting results allowed determination of the pseudo first-order rate constant for charge transfer (𝑘 &' ), expressed as the inverse of the 𝑅𝐶 time constant. The addition of [FeFe]-H2ase significantly enhances 𝑘 &' from 6.85 s -1 to 11.99 s -1 , whereas [NiFeSe]-H2ase shows a negative impact on 𝑘 &' with a value of 5.02 s -1 , further confirming the importance of specific interactions in facilitating the charge transfer process. The Bode phase plots (Figure ) revealed that the charge transfer process occurred within the frequency range of 0.1 Hz to 1 kHz, consistent with the reported timeframe for photocatalytic HER using CNX. Notably, the 𝑅𝐶 response of the conductive substrate forms a semicircle with a diameter of approximately 200 Ω in the Nyquist plots (Figure ) at high frequency region (10 kHz to 1 MHz, Figure ). To the best of our knowledge, this is the first impedance study conducted on CNX with H2ase, demonstrating specific interaction enables efficient DET by decreasing in 𝑅 &' and increasing in 𝑘 &' . In conclusion, we present a novel electrostatic strategy for linking enzymes with carbon nitrides, demonstrating a new benchmark for DET between NCN CNX and [FeFe]-H2ase for solar H2 production with a TON of 2×10 5 and a DET/MET ratio of 25% over 24 h. In contrast, the electrostatic repulsion between [NiFeSe]-H2ase and NCN CNX terminated DET, leading to a DET/MET ratio of 0.5%. QCM analysis demonstrates that specific interactions play a pivotal role in enabling DET, irrespective of the observed differences in the adsorption profiles. Additionally, PEIS analysis shows that interfacing [FeFe]-H2ase with NCN CNX facilitates charge transfer, as evidenced by a 23% less resistive 𝑅 &' and a 75% faster 𝑘 &' than bare NCN CNX. This study provides a promising and straightforward approach for achieving efficient electron transfer between carbon nitride and enzymes and serves as a reference for studying the charge carrier behavior of enzymephotocatalyst assemblies using interfacial characterizations.