[NiFe]-hydrogenases are highly efficient biological redox catalysts with the ability to interconvert H₂, protons, and electrons (H₂ ⇆ 2H⁺ + 2e⁻) at remarkable rates. Definition of the catalytic mechanism and particularly the parameters which make catalysis better in one reaction direction than in the other is important in both understanding microbial metabolism and in discovering and creating sustainable catalysts to underpin a H₂-energy economy. The Pyrococcus furiosus soluble [NiFe]-hydrogenases (PfSHI) is an oxygen-tolerant, group 3, soluble [NiFe]-hydrogenase, which grows optimally near 100 °C. This heterotetrameric enzyme consists of a heterdimeric [NiFe]-hydrogenase subcomplex and a dimeric NAD(P)-reducing diaphorase. Herein, the ability of PfSHI and its hydrogenase subdimer (PfSHI_H2ase) to catalyze H₂ production and oxidation electrocatalytically is studied using protein film electrochemistry. Electrochemical data show that both enzymes are active in hydrogen production and oxidation at low overpotentials. However, the activity of the two enzymes is not the same. The hydrogen subdimer is less active in H₂ oxidation under 100% H₂ atmosphere than the holoenzyme, but H₂ production activity under 100% N₂ atmosphere is unaffected by removal of the diaphorase subunits. Since absolute determination of turnover rates requires knowledge of electroactive coverage of enzyme on the electrode surface a parameter which is difficult to determine, the catalytic bias, i.e. the ratio of the maximal rates for hydrogen oxidation and proton reduction, was used to evaluate quantitatively the impact of mutation on activity. For PfSHI, the uneven response toward H₂ production and H₂ oxidation resulted in a dramatic shift in the catalytic biases which is calculated to be 7.1 in comparison with PfSHI_H2ase that is 1.4 under similar conditions. The influence of pH and temperature on the catalytic biases of PfSHI and PfSHI_H2ase was also evaluated. Both enzymes oxidize H₂ more efficiently under basic conditions, at pH 8.0, and reduce protons faster in acidic conditions, pH 5.5. The catalytic biases are also temperature dependent. For PfSHI, the rates of both oxidative and reductive catalysis increase with temperature. However, the rate of H₂ production increases much faster, resulting in a considerable decrease of the biases. The PfSHI_H2ase enzyme is marginally faster in the oxidative direction at low temperatures. However, the increase in temperature causes an uneven increase in catalytic H₂ production and H₂ oxidation rates which results in a shift in the catalytic bias to favor the proton reduction direction at elevated temperatures. However, the bias of PfSHI shifts more with temperature than the catalytic bias of PfSHI_H2ase. The shift in catalytic bias with temperature may indicate that there are different rate-determining steps for the forward and reverse reactions each with a distinct temperature dependence.