Baerbel Friedrich

Photosynthetic Hydrogen Production by a Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase

Nature provides key components for generating fuels from renewable resources in the form of enzymatic nanomachines which catalyze crucial steps in biological energy conversion, for example, the photosynthetic apparatus, which transforms solar power into chemical energy, and hydrogenases, capable of generating molecular hydrogen. As sunlight is usually used to synthesize carbohydrates, direct generation of hydrogen from light represents an exception in nature. On the molecular level, the crucial step for conversion of solar energy into H(2) lies in the efficient electronic coupling of photosystem I and hydrogenase. Here we show the stepwise assembly of a hybrid complex consisting of photosystem I and hydrogenase on a solid gold surface. This device gave rise to light-induced H(2) evolution. Hydrogen production is possible at far higher potential and thus lower energy compared to those of previously described (bio)nanoelectronic devices that did not employ the photosynthesis apparatus. The successful demonstration of efficient solar-to-hydrogen conversion may serve as a blueprint for the establishment of this system in a living organism with the paramount advantage of self-replication.

Characterization of the active site of a hydrogen sensor from Alcaligenes eutrophus

A third hydrogenase was recently identified in the proteobacterium Alcaligenes eutrophus as a constituent of a novel H2‐sensing multicomponent regulatory system. This regulatory hydrogenase (RH) has been overexpressed in cells deficient in both the NAD+‐reducing [NiFe]‐hydrogenase and the membrane‐bound [NiFe]‐hydrogenase. EPR, FTIR and activity studies of membrane‐free extracts revealed that the RH has an active site much like that of standard [NiFe]‐hydrogenases, i.e. a Ni‐Fe site with two CN− groups and one CO molecule. Its catalytic power is low, but the RH is always active, insensitive to oxygen, and occurs in only two redox states.

A hydrogen-sensing multiprotein complex controls aerobic hydrogen metabolism in Ralstonia eutropha.

H(2) is an attractive energy source for many microorganisms and is mostly consumed before it enters oxic habitats. Thus aerobic H(2)-oxidizing organisms receive H(2) only occasionally and in limited amounts. Metabolic adaptation requires a robust oxygen-tolerant hydrogenase enzyme system and special regulatory devices that enable the organism to respond rapidly to a changing supply of H(2). The proteobacterium Ralstonia eutropha strain H16 that harbours three [NiFe] hydrogenases perfectly meets these demands. The unusual biochemical and structural properties of the hydrogenases are described, including the strategies that confer O(2) tolerance to the NAD-reducing soluble hydrogenase and the H(2)-sensing regulatory hydrogenase. The regulatory hydrogenase that forms a complex with a histidine protein kinase recognizes H(2) in the environment and transmits the signal to a response regulator, which in turn controls transcription of the hydrogenase genes.

Transcriptional regulation of nitric oxide reduction in Ralstonia eutropha H16.

Nitric oxide reduction in Ralstonia eutropha H16 is catalysed by the quinol-dependent NO reductase NorB. norB and the adjacent norA form an operon that is controlled by the sigma(54)-dependent transcriptional activator NorR in response to NO. A NorR derivative containing MalE in place of the N-terminal domain binds to a 73 bp region upstream of norA that includes three copies of the putative upstream activator sequence GGT-(N(7))-ACC. Mutations altering individual bases of this sequence resulted in an 80-90% decrease in transcriptional activation by wild-type NorR. Similar motifs are present in several proteobacteria upstream of genes encoding proteins of NO metabolism. The N-terminal domain of NorR contains a GAF module and is hypothesized to interact with a signal molecule. A NorR derivative lacking this domain activates the norAB promoter constitutively. Amino acid exchanges within the GAF module identified a cysteine residue that is essential for promoter activation by NorR. Signal sensing by NorR is negatively modulated by the iron-containing protein NorA.

Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

Background: Biosynthesis of complex metal cofactors in [NiFe] hydrogenase is sensitive toward molecular oxygen. Results: A rubredoxin-like protein is required for hydrogenase maturation under aerobic conditions. Conclusion: The rubredoxin-like protein prevents oxidative damage of metallocenters, including the recently discovered [4Fe3S] center. Significance: Dedicated protection mechanisms enable biosynthesis of sophisticated metal centers in the presence of dioxygen. The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster.

Non-standard structures of the Ni-Fe cofactor in the regulatory and the NAD-reducing hydrogenases from Ralstonia eutropha.

Spectroscopy on two oxygen-insensitive Ni-Fe hydrogenases from Ralstonia eutropha (NAD-reducing, soluble hydrogenase; hydrogen sensor, regulatory hydrogenase) reveals non-standard catalytic behaviour and unique structures of their Ni-Fe cofactors. Possible mechanistic implications are briefly discussed.