ELECTRONIC STRUCTURE OF BIOMOLECULES

The application of Mossbauer spectroscopy to the investigation of biomolecules is discussed for heme proteins. All heme proteins have basically the same active center, namely heme. The heme iron is usually either ferric or ferrous ; in either oxidation state both high-spin and low-spin configurations can occur. The low temperature Mossbauer spectra can be described by a spin Hamiltonian of the form In low symmetries, which nature seems to prefer in biomolecules, the spin Hamiltonian will have a large number of unknown coefficients and the evaluation of the spectra is a formidable task. The Mossbauer spectra typical for the various charge and spin states of the heme iron are discussed ; cytochrome P450, chloroperoxidase and horseradish peroxidase are used as examples. For ferric heme proteins Epr results can be used to significantly facilitate the analysis of the Mossbauer spectra. For high-spin ferrous proteins Mossbauer measurements in strong applied fields are particularly useful ; such measurements on reduced cytochrome P450 are discussed in detail. In addition, MGssbauer emission experiments on 57C0 substituted hemes are presented ; this type of spectroscopy provides a means of studying heme model compounds which cannot be prepared chemically. 1 . Introduction. Nature abounds with a wide variety of iron containing biomolecules which challenge the talents of researchers in many fields. In addition to being involved in many of the fundamental processes that sustain life, these biomolecules offer the Mossbauer spectroscopist every degree of scientific challenge. In the past years the Mossbauer effect, which is the focus of this International Conference, has made a sizeable contribution to unveiling some unique biological structures. In this paper we will discuss Mossbauer spectroscopy of a special class of biomolecules the heme proteins. However, since much of the literature dealing with Mossbauer spectroscopy of biomolecules is scattered (*) New address : Department of BiochemistryIFreshwater Biological Institute, University of Minnesota, St. Paul, Minnesota 55101, USA. through physical and biological journals, we would briefly like to mention some structures which we will not discuss here but which are quite interesting from a stand-point of physics. There is the large class of so-called plant-type ferredoxins which are involved in electron transport in plant, bacterial or mammalian systems ; in addition they act as primary electron acceptors in photosynthesis. These proteins have a fascinating active site made up of two iron atoms which are spin-coupled via sulfur bridges. Mossbauer spectroscopy has played an indispensable role in revealing features of these unique structures [I, 21. Bacterial-type ferredoxins feature spin-coupled clusters involving four iron atoms situated a t alternate corners of a distorted cube. One protein, the ferredoxin from Chlostridium pasteurianum (a bacterium) has two such clusters ; X-ray crystallography has Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1974604 C6-34 E. MUNCK AND P. M. CHAMPION revealed that these clusters are spaced apart by 12 A [3]. There is experimental evidence that the reduced form of this protein presents us with a double spin-coupling problem ; each individual cluster is strongly spin-coupled to a spin system and both cIusters are weakly coupled to yield a singlet and a triplet state. The plot thickens when we consider the MoFe proteins from nitrogenase, an enzyme system capable of reducing molecular nitrogen to ammonia. These proteins contain two molybdenum and some twentyodd iron atoms. Recently these proteins have been studied by the Mossbauer effect [4] and by Epr spectroscopy and it appears that nature utilizes yet another type of cluster, containing (probably) four iron atoms, spin-coupled to a total spin of S = 3 (E. Miinck et al., submitted for publication). The MoFe protein seems to have two such clusters accounting for a total of 8 iron atoms. The other irons reside in complexes of integer electronic spin and are therefore not amenable to most of the spectroscopic methods. Thus the elucidation of structural details of the MoFe proteins falls squarely upon the shoulders of Mossbauer spectroscopy. In this paper we shall be concerned with heme proteins. These proteins, immensely important in biological systems, serve as good examples to show what information can be obtained from Mossbauer spectroscopy. Elegant reviews about the application of Mossbauer spectroscopy to biological materials have been written by Lang [5 ] and Debrunner [6]. 2. Cytochrome P450 : an example. Heme proteins perform many vital functions in biological systems. Probably the best known example of this class is hemoglobin, the oxygen carrier in the blood. All heme proteins have basically the same prosthetic group, namely Fe-protoporphyrin IX (heme). As depicted in figure 1, the iron atom is incorporated into an aromatic ring and coordinated to four nitrogen atoms. In general, a fifth (axial) coordination position is occupied by an amino acid residue (like histidine in hemoglobin) ; the sixth position is either vacant, or occupied by another amino acid residue or by an extraneous ligand (like O,, CO, etc.). This multitude of possible coordinations permits the amazing diversity of biological functions that characterize the families of heme proteins (electron transport, oxygen transport, and a variety of catalytic reactions). From a standpoint of Mossbauer spectroscopy the variability in stereochemica1 coordination results in different electronic spin configurations which give their characteristic signature in the Mossbauer spectra. As an example we discuss cytochrome P450, an enzyme which catalyses the hydroxylation of camphor. This protein has a molecular weight of 40 000 (393 amino acids) and has one heme group. The axial ligands to the heme iron are not known with certainty, though cysteine is suspected. In the following sections FIG. 1. The heme group (Fe-protoporphyrin IX). Eight side chains are attached to the periphery of the porphyrin ring (4 methyl, 2 vinyl and 2 propionic acid groups). we will discuss some of the Mossbauer data which have been obtained for this protein. Cytochrome P450 has been isolated from a bacterium, Pseudomonas putida, by Gunsalus and coworkers [7]. The proposed reaction mechanism is shown in figure 2. We start at the lower left in figure 2 and move clockwise around the