Protein film electrochemistry (PFE) was utilized to characterize the catalytic activity and oxidative inactivation of a bidirectional [NiFe]-hydrogenase (HoxEFUYH) from the cyanobacterium Synechocystis sp. PCC 6803. PFE provides precise control of the redox potential of the adsorbed enzyme so that its activity can be monitored under changing experimental conditions as current. The properties of HoxEFUYH are different from those of both the standard uptake and the "oxygen-tolerant" [NiFe]-hydrogenases. First, HoxEFUYH is biased toward proton reduction as opposed to hydrogen oxidation. Second, despite being expressed under aerobic conditions in vivo, HoxEFUYH is clearly not oxygen-tolerant. Aerobic inactivation of catalytic hydrogen oxidation by HoxEFUYH is total and nearly instantaneous, producing two inactive states. However, unlike the Ni-A and Ni-B inactive states of standard [NiFe]-hydrogenases, both of these states are quickly (<90 s) reactivated by removal of oxygen and exposure to reducing conditions. Third, proton reduction continues at 25-50% of the maximal rate in the presence of 1% oxygen. Whereas most previously characterized [NiFe]-hydrogenases seem to be preferential hydrogen oxidizing catalysts, the cyanobacterial enzyme works effectively in both directions. This unusual catalytic bias as well as the ability to be quickly reactivated may be essential to fulfilling the physiological role in cyanobacteria, organisms expected to experience swings in cellular reduction potential as they switch between aerobic conditions in the light and dark anaerobic conditions. Our results suggest that the uptake [NiFe]-hydrogenases alone are not representative of the catalytic diversity of [NiFe]-hydrogenases, and the bidirectional heteromultimeric enzymes may serve as valuable models to understand the diverse mechanisms of tuning the reactivity of the hydrogen activating site.

Light-harvesting antenna complexes not only aid in the capture of solar energy for photosynthesis, but regulate the quantity of transferred energy as well. Light-harvesting regulation is important for protecting reaction center complexes from overexcitation, generation of reactive oxygen species, and metabolic overload. Usually, this regulation is controlled by the association of lightharvesting antennas with accessory quenchers such as carotenoids. One antenna complex, the Fenna-Matthews-Olson (FMO) antenna protein from green sulfur bacteria, completely lacks carotenoids and other known accessory quenchers. Nonetheless, the FMO protein is able to quench energy transfer in aerobic conditions effectively, indicating a previously unidentified type of regulatory mechanism. Through de novo sequencing MS, chemical modification, and mutagenesis, we have pinpointed the source of the quenching action to cysteine residues (Cys49 and Cys353) situated near two lowenergy bacteriochlorophylls in the FMO protein from Chlorobaculum tepidum. Removal of these cysteines (particularly removal of the completely conserved Cys353) through N-ethylmaleimide modification or mutagenesis to alanine abolishes the aerobic quenching effect. Electrochemical analysis and electron paramagnetic resonance spectra suggest that in aerobic conditions the cysteine thiols are converted to thiyl radicals which then are capable of quenching bacteriochlorophyll excited states through electron transfer photochemistry. This simple mechanism has implications for the design of bio-inspired light-harvesting antennas and the redesign of natural photosynthetic systems.photosynthesis | Fenna-Matthews-Olson protein | excitation quenching | thiyl radical | light-harvesting P hotosynthesis can be performed in the presence of oxygen (oxygenic photosynthesis, in which water is the electron source) or in its absence (anoxygenic photosynthesis, in which other reduced species such as sulfide are the electron sources) (1-3). Many bacteria performing anoxygenic photosynthesis contain type I reaction center (RC) protein complexes that use Fe-S clusters to transfer electrons to ferredoxin or related molecules after photoinduced charge separation (4-6). These Fe-S clusters are easily damaged by molecular oxygen. Therefore, anoxygenic phototrophs must use a pathway either to remove oxygen or to reduce the rate of photosynthesis whenever oxygen is encountered (1, 5).Phototrophic members of the bacterial phylum Chlorobi (green sulfur bacteria, GSBs) are anoxygenic phototrophs that contain such a type I RC. They also contain two peripheral antenna complexes that aid in regulating light absorption and energy transfer: the chlorosome and the homotrimeric Fenna-Matthews-Olson (FMO) protein complex. When oxygen is encountered, the bacteria are able to decrease the output of photosynthesis. This process involves creating trapping sites in the antenna complexes where de-excitation processes outcompete the rate of energy transfer to the RC, preventing RC damage. This process is well un...

Stable immobilization of two redox proteins, cytochrome c and azurin, in a thin film of highly mesoporous antimony-doped tin oxide is demonstrated via UV-vis spectroscopic and electrochemical investigation.

We report the first direct electrochemical characterization of the impact of oxygen on the hydrogen oxidation activity of an oxygen-tolerant, group 3, soluble [NiFe]-hydrogenase: hydrogenase I from Pyrococcus furiosus (PfSHI), which grows optimally near 100 °C. Chronoamperometric experiments were used to probe the sensitivity of PfSHI hydrogen oxidation activity to both brief and prolonged exposure to oxygen. For experiments between 15 and 80 °C, following short (<200 s) exposure to 14 μM O2 under oxidizing conditions, PfSHI always maintains some fraction of its initial hydrogen oxidation activity; i.e., it is oxygen-tolerant. Reactivation experiments show that two inactive states are formed by interaction with oxygen and both can be quickly (<150 s) reactivated. Analogous experiments, in which the interval of oxygen exposure is extended to 900 s, reveal that the response is highly temperature-dependent. At 25 °C, under sustained 1% O2/ 99% H2 exposure, the H2oxidation activity drops nearly to zero. However, at 80 °C, up to 32% of the enzyme's oxidation activity is retained. Reactivation of PfSHI following sustained exposure to oxygen occurs on a much longer time scale (tens of minutes), suggesting that a third inactive species predominates under these conditions. These results stand in contrast to the properties of oxygen-tolerant, group 1 [NiFe]-hydrogenases, which form a single state upon reaction with oxygen, and we propose that this new type of hydrogenase should be referred to as oxygen-resilient. Furthermore, PfSHI, like other group 3 [NiFe]-hydrogenases, does not possess the proximal [4Fe3S] cluster associated with the oxygen tolerance of some group 1 enzymes. Thus, a new mechanism is necessary to explain the observed oxygen tolerance in soluble, group 3 [NiFe]-hydrogenases, and we present a model integrating both electrochemical and spectroscopic results to define the relationships of these inactive states.

The majority of protein spectroelectrochemical methods utilize a diffusing, chemical mediator to exchange electrons between the electrode and the protein. In such methods, electrochemical potential control is limited by mediator choice and its ability to interact with the protein of interest. We report an approach for unmediated, protein spectroelectrochemistry that overcomes this limitation by adsorbing protein directly to thiol self-assembled monolayer (SAM) modified, thin (10 nm), semitransparent gold. The viability of the method is demonstrated with two diverse and important redox proteins: cytochrome c and azurin. Fast, reversible electrochemical signals comparable to those previously reported for these proteins on ordinary disk gold electrodes were observed. Although the quantity of protein in a submonolayer adsorbed at an electrode is expected to be insufficient for detection of UV-vis absorption bands based on bulk extinction coefficients, excellent spectra were detected for each of the proteins in the adsorbed state. Furthermore, AFM imaging confirmed that only a single layer of protein was adsorbed to the electrode. We hypothesize that interaction of the relatively broad gold surface plasmon with the proteins' electronic transitions results in surface signal enhancement of the molecular transitions of between 8 and 112 times, allowing detection of the proteins at much lower than expected concentrations. Since many other proteins are known to interact with gold SAMs and the technical requirements for implementation of these experiments are simple, this approach is expected to be very generally applicable to exploring mechanisms of redox proteins and enzymes as well as development of sensors and other redox protein based applications.