ARTICLE E. Balog á R. GalaÂntai á M. KoÈhler M. Laberge á J. Fidy Metal coordination in¯uences substrate binding in horseradish peroxidase Received: 15 October 1999 / Revised version: 3 April 2000 / Accepted: 5 April 2000 Abstract To clarify the role of metal ion coordination in horseradish peroxidase C HRPC), the eect of pressure and of an externally applied electric ®eld on spectral holes was compared for both metal-free and Mg-meso- porphyrin-substituted horseradish peroxidase C MP- HRP and MgMP-HRP), as aected by the binding of 2-naphthohydroxamic acid NHA). The data are compared to earlier studies performed on the same de- rivatives. Results obtained for MP-HRP show the presence of a predominant MP tautomer, as well as that of another small population with dierent pocket ®eld and isothermal compressibility 0.12 vs 0.24 GPa )1 ). Binding NHA induces the formation of two new almost equal populations of MP-HRP tautomer complexes and the protein compressibility in both forms is increased to 0.50 and 0.36 GPa )1 . The protein structure becomes much softer than in the absence of NHA. Binding the same substrate to MgMP-HRP resulted in MgMP adopting a single conformation with no compressibility changes, while without NHA, two forms were possible. Stark eect results show charge rearrangement upon substrate binding in both cases. We propose that it is the presence of the metal that stabilizes the structure during the reorganization of the protein matrix induced by the substrate binding event. With the metal, only one conformation is adopted, without signi®cant structural rearrangement but with charge redistribution. The dis- sociation constants determined for NHA binding to both derivatives and to native HRPC show that studies using mesoporphyrin and Mg-mesoporphyrin deriva- tives are relevant to investigating the speci®city of the substrate-binding pocket in this enzyme. Key words Horseradish peroxidase á Spectral hole burning á Protein isothermal compressibility á Stark eect á Porphyrin Q-band splitting Abbreviations HRPC isozyme C of horseradish pero- xidase á MP mesoporphyrin IX á MP-HRP mesopor- phyrin IX horseradish peroxidase C á MgMP á MgII)- mesoporphyrin IX á MgMP-HRP MgII)-mesopor- phyrin IX horseradish peroxidase C á NHA 2- naphthohydroxamic acid á SHB spectral hole burning Introduction We have been studying the C isozyme of horseradish peroxidase HRPC) for a long time. It contains a non- covalently bound heme group maintained in the protein matrix by a network of hydrophobic and electrostatic interactions Yonetani et al. 1972). Our interest is not only motivated by the fact that it is representative of the important biochemical class of peroxidases, but also because it is a good model system to investigate the role of protein conformation in stabilizing a given ± func- tionally important ± structure Fidy et al. 1998; Fried- rich et al. 1994; Herenyi et al. 1995). HRPC is a good candidate because it binds such a wide variety of sub- strates that it is considered to defy all rules of enzyme speci®city Hewson and Hager 1979). As such, it then provides us with a protein system expected to undergo a variety of structural rearrangements to accommodate its dierent substrates Fidy et al. 1989, 1992a). Speci®cally, we are interested in assessing the role ± and the inter- actions ± of the dierent elements making up the enzyme assembly. In a simple approach, we may consider the porphyrin prosthetic group with a central metal, em- bedded in a protein matrix that may acquire speci®c functionally signi®cant populations of substates as a consequence of interactions with the porphyrin. The question is what interactions are important in this selection. Eur Biophys J 2000) 29: 429±438 Ó Springer-Verlag 2000 E. Balog á R. GalaÂntai á M. Laberge á J. Fidy &) Institute of Biophysics, Semmelweis University of Medicine, P.O. Box 2634, 1444 Budapest, Hungary e-mail: judit@puskin.sote.hu M. KoÈ hler Technische UniversitaÈt MuÈnchen, Lehrstuhl fuÈr Physik Weihenstephan, 85350 Freising, Germany