STRUCTURE AND FUNCTION OF REDOX METALLOPROTEINS

1. Structure and function of redox proteins

Redox proteins play an important role in the bio-energetic processes of living organisms. Energy producing, utilizing, transforming processes are present in all living systems; the research of redox proteins can hardly be overestimated. Most of the redox proteins contain some kind of metal (iron, copper, etc.).

Our group isolates proteins from the purple, sulphur, photosynthetic organism Thiocapsa roseopersicina. Several metal-containing and redox active proteins have already been purified. The long-time favourite and most widely investigated protein is Hyn hydrogenase (see next section). We have also identified and characterized a number of other metal-containing proteins, including a flavocytochrome, a cytochrome c4 (yet unknown from photosynthetic organism) and a blue copper protein. We have determined their characteristics (redox potential, molecular mass, etc.). Despite the fact that the organism does not survive temperatures above 30 °C, the cytochrome c4 is stable and functional at high temperatures (up to 60 °C). The protein undergoes conformational changes upon increases in temperature. These conformational changes are reversible under anaerobic conditions and irreversible if oxygen is present.

Development and exploitation of the results

The investigation of these proteins helps to understand the temperature tolerance of the proteins. On the other hand the techniques developed in the course of research are also important and can be used for other research. The most important technique is a new protein-sequencing methodology, which was developed in cooperation with an American MS group in Texas. The protein sequence is determined with the help of a mass spectrometer. Usually the protein sequence is determined by determining the nucleic acid sequence and translating it to protein sequence. It is a quite time-consuming, complicated and not always successful method for genetically unmapped organisms (and most organisms belong to this group). Furthermore the sequence does not necessarily fit the sequence of the working protein. Our new method is competitive with sequence determination from the gene, and we hope that with some improvement it will be possible to decrease the required amount of protein to the level of two-dimensional gel spots. The drawback of this method is that it needs expensive mass spectrometers.

2. Investigation of autocatalytic and oscillating enzyme reactions

Autocatalytic reactions are quite common in living systems; they are very easy to observe macroscopically. Proliferation and reproduction are typical autocatalytic processes. In order to produce posteriors, in addition to food one or two parents are necessary.

The autocatalytic reaction of hydrogenase
in thin layer

Autocatalytic processes are easily recognized by their characteristic spatial patterns. In the absence of other disturbing effects the reaction fronts are spherical (or, in the case of flat reaction arrangements, circular). The radii of the objects are continuously increasing in time (see the figure). The spread of humankind on Earth has a very similar pattern.

Although autocatalytic reactions are common in living systems, they are quite rare in elementary reactions. We investigate such elementary reactions on bio-molecules and enzymes.

The main object of our study is the hydrogenase enzyme. It can be found mostly in prokaryotes and archae, but some eukaryotes might contain it as well.

Hydrogenase catalyzes a very simple reaction: it splits hydrogen gas into protons and electrons and, as all catalysts, it works in the reverse direction as well, i.e. it produces hydrogen gas from electrons and protons. The enzyme contains metal cofactors, iron, and some hydrogenases also contain nickel. We have found that during the enzyme reaction there is at least one autocatalytic step involved.

Development and exploitation of the results

Autocatalytic reactions are also present in other biological processes. The development of "prion" diseases (Creutzfeldt-Jakob disease, scrapie, bovine spongiform encephalopathy, etc.) is also described as an autocatalytic process. In the case of prion proteins, however, the investigation of the kinetics of the autocatalytic reaction (how the reaction proceeds, what happens during the reaction) is very difficult. Since the reaction of hydrogenase is easy to follow and the concentration of different components can easily be changed, there is hope that the information and the techniques developed in research on this reaction can be applied to the case of prion reactions.

The autocatalytic reaction of hydrogenase has biotechnological importance. We do not think that with the help of hydrogenase bio-hydrogen would be produced in industrial quantity by splitting the water. But we do think that hydrogenase can be utilized as a key constituent of fuel cells. It might substitute the expensive platinum, because it is cheaper, and it doesn’t have to be mined, because it can be produced utilizing solar energy. For its utilization, however, it is essential to precisely know the reaction kinetics.