Metal coordination influences substrate binding in horseradish peroxidase

Abstract

To clarify the role of metal ion coordination in horseradish peroxidase C (HRPC), the effect of pressure and of an externally applied electric field on spectral holes was compared for both metal-free and Mg-mesoporphyrin-substituted horseradish peroxidase C (MP-HRP and MgMP-HRP), as affected by the binding of 2-naphthohydroxamic acid (NHA). The data are compared to earlier studies performed on the same derivatives. Results obtained for MP-HRP show the presence of a predominant MP tautomer, as well as that of another small population with different pocket field 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 effect 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 significant structural rearrangement but with charge redistribution. The dissociation constants determined for NHA binding to both derivatives and to native HRPC show that studies using mesoporphyrin and Mg-mesoporphyrin derivatives are relevant to investigating the specificity of the substrate-binding pocket in this enzyme.

Protein matrix local fluctuations and substrate binding in HRPC: A proposed dynamic electrostatic sampling method

“…Necessarily, we need to extend the analysis to other protein matrix regions. However, we are able to show that: (1) R‐chain rearrangements do occur as a result of the substrate‐binding event, (2) the largest deviations between the free and substrate‐bound structures are observed in the residues that are recognized to have catalytic importance, and (3) the dynamics of the side‐chains of these residues also show fluctuations in their nonbond energies, which translate into a fluctuating electrostatic potential in the binding region—the effect of which we could show experimental evidence for in previous work 23, 24. Further work will hopefully elucidate the quantitative nature of these field fluctuations: they need to investigate all binding channels identified so far in the peroxidase, and report on other properties, which also deserve careful consideration, such as the fluctuations in the planarity of the heme macrocycle and the coupling to larger amplitude matrix motions.…”

“…We elected to model the fluctuations of both native HRPC and its Mg‐substituted analog because we are interested in studying the role of the central metal in HRP 24, and are presently assessing QM/MM methods for this purpose 35. It was felt that an MM comparison of the two derivatives would provide us with a benchmark for the further development of an appropriate QM/MM method—required to model an open‐shell metal.…”

“…In these studies, recently reviewed 21, 22, we used high‐resolution laser spectroscopy methods, such as Stark effect spectral hole burning and fluorescence line narrowing, to study the effect of substrate binding on the spectral properties of the prosthetic group of a modified enzyme, Mg (II)–mesoporphyrin IX horseradish peroxidase C (MgMP–HRP). In our most recent studies 23, 24, we were able to obtain direct experimental evidence for charge redistribution in the protein matrix around the porphyrin active site, and accordingly felt it of interest to further investigate substrate specificity by modifying a rough electrostatic conformational searching method previously used to sample dynamic field heterogeneity in c‐type cytochromes 25. We summarize elsewhere the general problem of modeling the electrostatics of complex biophysical systems 26, and refer to recent simulations of electrostatic substrate steering which convincingly show that electrostatic steering is not only the result of contributions from charged R‐groups, but rather from the entire partial charge distribution of the protein 14, 27, 28.…”

The charge transfer band in horseradish peroxidase correlates with heme in‐plane distortions induced by calcium removal

“…The large Q v envelope at 300 K also includes the buried Q 0 component of the HS/QS species. In the 10 K spectra, we can resolve the respective Q 0 components of the HS/QS state and of the LS state, each with respective Q v separated by some 1300 cm −1 , well within the range of observed Q v ‐Q 0 separations,16 as well as a split CT band. At 300 K, the Ca 2+ ‐free sample yields a spectrum with increased LS component at 540 nm (red trace) with a much decreased HS/QS component.…”

In vivoproduction of catalase containing haem analogues

“…The haem group of some water‐soluble proteins can, in vitro , be removed and reinserted, or substituted for another metalloporphyrin, such as Zn‐PP or Co‐PP [24]. The assembly of more complex haem proteins, such as membrane‐bound respiratory enzymes or catalase, however, can, at present, generally not be accomplished in vitro .…”