Substrate binding triggers a switch in the iron coordination in dehaloperoxidase from Amphitrite ornata: HYSCORE experiments.

We have explored the effect of substrate binding on the heme iron conformation in the enzyme dehaloperoxidase (DHP) that was first isolated from the terebellid polychaete Amphitrite ornata and is now expressed in Escherichia coli.1-3 DHP is a dimeric hemoglobin4 that also has significant peroxidase activity under physiological conditions.5 Since hemoglobins and peroxidases require ferrous and ferric oxygen, respectively, one can hypothesize that substrate binding causes a change in protein conformation that affects the spin state of the heme iron. A recent X-ray crystal structure of metaquo DHP resting state6 shows that water is indeed bound to the heme iron as predicted by spectroscopic measurements.7-9 In attempting to understand the role of substrate binding, we obtained an indication from hyperfine-shifted NMR that the substrate enters the distal pocket at pH < 7.0. At pH < 7.0, the enzyme turnover is rapid and so is the inactivation of DHP.2,3 Above pH 7.0, the turnover is significantly slower, but substantially more product is formed. Although an X-ray crystal structure10 shows a substrate analogue, 4-iodophenol, to be bound in the distal pocket of the hemoglobin, stopped-flow experiments (not shown) indicate that DHP can function without the substrate bound to the internal binding site. The ∼23% occupancy of the substrate analogue 4-iodophenol in the X-ray structure 1EWA10 suggests a function for the substrate interaction in the distal pocket but is inconclusive whether the distal pocket is an active site of the enzyme. We have advanced a hypothesis that substrate binding acts as a trigger event in the switch from the oxygen binding to the peroxidase function.1-3 An experimental test correlating changes in the ligation state with the coordination of the ferric heme iron upon substrate binding is considered to be critical for verification of our hypothesis. Herein we report on continuous wave (CW) EPR and hyperfine sublevel correlation spectroscopic (HYSCORE) analysis of the ferric form of DHP that was undertaken to characterize effects of the binding of 2,4,6-trifluorophenol (TFP) on heme iron coordination. HYSCORE experiments that correlate nuclear frequencies in the two manifolds of the electronic spin are informative for studying the hyperfine interactions of the heme iron with the surrounding nuclei. Experimental X-band (9.5 GHz) CW EPR spectra of DHP in the absence (dotted line) and the presence (solid line) of a 10-fold excess of TFP at pH 6.0 are shown in Figure 1. The characteristic g⊥) 6 and g|| ) 2 features of the EPR spectra show that the iron exists in a high spin (HS, S ) /2) state in both the absence and presence of the substrate.14,19 In this respect, DHP resembles metmyoglobin, a known HS ferric heme protein having a sixcoordinate ligation. At pH 6.0, there is no change in the spin state; however, the coordination sphere of the heme iron is clearly affected by substrate binding (Figure 1 inset). Figure 2 shows HYSCORE spectra of DHP at pH ) 6.0 in an H2O buffer (A), a D2O (99%) buffer (B), and an H2O buffer with 10-fold excess of TFP relative to DHP (C). The spectrum of DHP in the second quadrant reveals the signals from strongly coupled nitrogen nuclei at (-9.5, 5.54) MHz that are assigned to a double quantum (∆mI ) (2, dq) transition. Two less intense peaks at (-4.98, 2.95) and (-4.45, 3.15) MHz arise from single quantum (∆mI ) (1, sq) transitions. These signals were assigned to four approximately equivalent nitrogen nuclei of the porphyrin ring and another nitrogen of the proximal His89.11,12 The first quadrant revealed proton signals at 14.8 MHz that span about 6.2 MHz frequency range with a well-defined strong intensity characterized by weaker interactions of 2.5 MHz and lower. To better understand the origin of the proton signals, a spectrum of DHP prepared in pH 6.0 D2O buffer was obtained (Figure 2B). For this sample preparation, all of the spectral features in both quadrants remained the same, except the signals corresponding to strongly coupled (6 MHz) proton(s) that disappeared. This indicates that the signal from the weakly coupled proton(s) with interaction of about 2.5 MHz originates from nonexchangeable protein protons. Hyperfine interactions of similar magnitude have been observed for hydrogen atoms in the heme and proximal histidine.13,14 The disappearance of the strongly coupled 6 MHz proton signal in D2O buffer (Figure 2B) is attributed to exchangeable hydrogen atom(s). Previous ENDOR studies of metmyoglobin reported a 6.1 ( 0.1 MHz hyperfine coupling for the protons of a water molecule coordinated to the iron as the sixth ligand,15,16 which is essentially the same as the 6 MHz signal observed for DHP. Following buffer exchange, we have observed an additional intensity in the first quadrant at (1.83, 2.63) MHz that is consistent with the deuteron signals (νD ) 2.3 MHz). Unfortunately, for DHP, in both H2O and D2O buffers, this quadrant contains a strong spectral feature, extending from 1.5 to 3.8 MHz that prohibits unambiguous † North Carolina State University. ‡ Bruker BioSpin Corporation. Figure 1. CW X-band (9.5 GHz) EPR spectra of DHP in the absence (dotted line) and in the presence (solid line) of 10-fold excess of TFP at pH 6.0 and T ) 4 K. Arrow indicates the field of HYSCORE experiment. Published on Web 01/25/2008