Helmut Beinert

Engineering of protein bound iron‐sulfur clusters

An increasing number of iron-sulfur (Fe-S) proteins are found in which the Fe-S cluster is not involved in net electron transfer, as it is in the majority of Fe-S proteins. Most of the former are (de)hydratases, of which the most extensively studied is aconitase. Approaches are described and discussed by which the Fe-S cluster of this enzyme could be brought into states of different structure, ligation, oxidation and isotope composition. The species, so obtained, provided the basis for spectroscopic and chemical investigations. Results from studies by protein chemistry, EPR, Mössbauer, 1H, 2H and 57Fe electron-nuclear double resonance spectroscopy are described. Conclusions, which bear on the electronic structure of the Fe-S cluster, enzyme-substrate interaction and the enzymatic mechanism, were derived from a synopsis of the recent work described here and of previous contributions from several laboratories. These conclusions are discussed and summarized in a final section.

Copper in biological systems. A report from the 6th Manziana conference, September 23–27, 1990

Enzymes and proteins: AO, amine oxidase; and as proposed in reference 3, BSAO, bovine serum AO; SSAO, swine serum AO; SKDAO, swine kidney AO; PSAO, pea seedling AO; APAO, arthrobacter P1AO; MADH, methylamine dehydrogenase; AAO, ascorbic acid oxidase; alpha-AE, alpha-amidating enzyme; Az, azurin; COX, cytochrome c oxidase; CP, ceruloplasmin; DBH, dopamine beta-hydroxylase; GO, galactose oxidase; Hc, hemocyanin; MT, metallotheonein; NIR, nitrite reductase; SOD, superoxide dismutase. Cofactors: Dopa, 3,4 dihydroxyphenylalanine; Topa, 3,4,6 trihydroxyphenyl-alanine; PLP, pyridoxal-phosphate; PQQ, pyrroloquinolinequinone. Reagents: DDC, diethyldithiocarbamate; DMG, diaminoguanidine; DMSA, dimercaptosuccinic acid; NTA, nitrilotriacetic acid. Technique-related: XANES, x-ray absorption near edge spectroscopy; EXAFS, extended x-ray absorption fine structure; ENDOR, electron-nuclear double resonance; ESEEM, electron spin echo envelope modulation; CD, circular dichroism; MCD, magnetic circular dichroism; NMRD, nuclear magnetic resonance dispersion; nqi, nuclear quadrupole interaction; DSC, differential scanning calorimetry.

Heterogeneity in an isolated membrane protein Has the ‘authentic cytochrome oxidase’ been identified?

The criteria of homogeneity or native state of a protein are prone to become ambiguous when applied to membrane proteins, such as cytochrome‐c oxidase, which are purified by extraction with detergents. Properties of the purified material depend on the detergent used and on details of the purification protocol followed with any single batch of a preparation. We present arguments to show that the evidence presently available in published form does not justify the designation [(1987) J. Biol. Chem. 262 3160–3164] of one type of preparation as being closer to the native state than others.

The Iron Responsive Element (IRE), the Iron Regulatory Protein (IRP), and Cytosolic Aconitase

The fundamental role of iron in the maintenance of human health has been apparent for many years. The requirement for iron in growth and development of organisms from bacteria to humans arises because it is an essential component of proteins that perform redox and nonredox roles in a number of cellular functions. Given that iron is one of the most abundant, chemically versatile, and reactive elements in our environment, it is not surprising that nature has made extensive use of its properties. However, there are two significant problems regarding the use of iron in biological systems: its low solubility, particularly as Fe(III), and its toxicity when present in excess because of its ability to induce formation of damaging free radicals. Organisms have developed a variety of mechanisms to acquire and make use of iron for a large number of necessary functions while simultaneously reducing the incidence of inappropriate effects of this micronutrient on cell viability. Recent investigations of the regulation of iron homeostasis in mammals have identified two unique proteins: the iron regulatory proteins or IRPs,1 which act as central regulators of iron utilization. IRPs appear to represent the only members of the aconitase family of proteins that function in gene regulation (Frishman and Hentze 1996; Rouault et al. 1992). IRPs are cytosolic RNA binding proteins that modulate synthesis of proteins that function in the uptake, storage, and utilization of iron by binding to their mRNAs, thereby affecting their translation or stability. Posttranslational regulation of IRP function by iron and phosphorylation, with subsequent effects on iron metabolism, are topics of current inquiry to those interested in posttranscriptional gene regulation, iron-sulfur protein structure and function, and regulation of iron homeostasis.

Trails of inquiry and thought leading toward today's bioenergetics

Significant experimental observations and concepts leading from various sources toward todays bioenergetics are briefly sketched. To limit the essay to manageable proportions main consideration is given to origins in research on metabolism and biological oxidations and attendant energy conversions. Relevant data and dates are summarized in table form.

What do we and what don't we know today about cytochrome c oxidase? Overviews and summaries at the Accademia dei Lincei and discussion meeting of Caprarola.

In the footsteps and the spirit of the 1984 meeting’ a t the Accademia dei Lincei and the Casa San Teresa at Caprarola, our colleagues a t Rome organized similar successive meetings in June 1988 a t the same locations. While the center of the city of Rome, particularly in this season, has become a noisy and bewildering place, the palazzi and their walled-in gardens still provide islands of tranquility as did the park of the Villa Farnesina, where the Accademia dei Lincei is located. The meeting there was opened by Prof. Rossi-Fanelli, the doyen of heme protein research in Italy. The lectures a t the Accademia were partly overviews of or introductions to certain aspects of cytochrome oxidase structure, function, and physiopathology, and partly they presented recent progress and results of both experimental and conceptual nature. While the lectures of R. A. Capaldi, H. B. Gray, G. F. Azzone, S. Di Mauro, and B. Chance may be classified among the former, the remainder of the lectures dealt with not yet or only preliminarily published findings and views. Both A. Azzi’s and J. A. Fee’s papers on microbial oxidases supported the view that all four redox-active metal components (two hemes, Cu, and Cu,) can be, and in these oxidases apparently are present in subunit I. In a scholarly lecture G. Palmer presented an in-depth analysis of the conditions to be satisfied if one wants to arrive at a correct steady-state kinetic mechanism for the total reaction of an enzyme as complex as cytochrome c oxidase, a frightening picture indeed. M. T. Wilson’s lecture dealt with an analysis of conformational states and distance relationships between the redox components of the cytochrome c-cytochrome oxidase system, largely deduced via fluorescence measurements. Orii’s lecture showed the most detailed analysis yet performed on the reoxidation at ambient temperature of reduced oxidase by oxygen as observed by transient kinetic and optical absorption methods. In a session exclusively concerned with proton translocation by cytochrome oxidase, S. I . Chan, M. K. F. Wikstrom, P. Mitchell, B. G . Malmstrom, and G. F. Azzone spoke on presently discussed and, in several instances, opposing views of the detailed mechanism involved and regulation of the process. M. Brunori’s lecture, given the previous day, addressed the same topic. The degree of specific detail in which this topic is now being considered is impressive, but it is also obvious that more of an experimental foothold is required to elucidate the mechanism employed by nature. The biosynthesis of subunits and genetic aspects were discussed by R. P. Poyton on yeast and R. Bisson on slime molds. S. DiMauro and B. Chance led the audience beyond biochemical and genetic considerations into the rapidly expanding realm of pathological and physiological implications of cytochrome oxidase function or lack of function in lectures that clearly showed that practical application of the knowledge

Localized Valence States in Iron-Sulfur Clusters and Their Possible Relationship to the Ability of Iron-Sulfur Clusters to Catalyze Reactions other than Electron Transfer

The last few years have seen great progress in our knowledge of the large variety and wide distribution of iron-sulfur (Fe-S) clusters as biocatalysts. A priori one might predict that a structure made up of four atoms of a transition metal that can be found in several stable valency and spin states plus four atoms of a “soft” chalcogen that can occur in stable ionic forms with oxidation states 2− to 6+ should yield a device of remarkable properties. We are learning that this is indeed the case, particularly for the larger, i.e. 4Fe clusters. They are surprisingly malleable entities which readily respond to subtle influences from the surrounding ligands furnished by the protein and (or) from additional ligands approaching from the medium. Intuitively, these would seem exactly conditions required for producing a potential active site of an enzyme and there is evidence that nature indeed makes use of these circumstances.