A Photolysis-Triggered Heme Ligand Switch in H93G Myoglobin

Abstract

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116,[6981][6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in <5 µs. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.

Proximal ligand motions in H93G myoglobin

“…3 indicate that the axial ligand of the heme iron is not the same in the H93G-CO and deoxy H93G species. This is in agreement with step-scan FTIR and saturation Raman data that indicate a dynamic ligand switch in H93G Mb [32]. The histidine that appears bound in the 8 ns photoproduct spectrum dissociates from the iron on a time scale < 5 ls.…”

“…This is certainly the case for H93G-*CO shown in Fig. 3, as it is known that a ligand switch occurs in H93G-*CO [32]. Of the ligands used in this study, it is likely that 2-methyl imidazole dissociates the most rapidly given the steric hindrance between the 2-methyl group of this ligand and the heme.…”

“…Additionally, data are presented for the proximal cavity mutant in the absence of exogenous ligand (H93G). In this case the axial ligand trans to CO is one of the histidine residues located in the heme pocket of the globin [32]. Experimental comparison of the deoxy form for a ligand L [denoted H93G(L)] and the photolyzed CO form [denoted H93G(L)*CO] permits a comparison of the equilibrium deoxy state with that of a nonequilibrium state very close to that of the ligated species.…”

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

“…These proteins, like some mutant hemoproteins with a tyrosinate as single axial ligand, have I( 4 )/I( 3 ) intensity ratios in the 1-1.5 range. Such intensity ratios have also been reported for 5cHS hemoproteins with a coordinated hydroxide ion (32,33). No isotope shift was observed for H32A in H 2 18 O at pH 10.0 indicating that a hydroxide complex was not formed and that Tyr 75 was the most likely anionic oxygen heme ligand.…”

Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

“…Upon reduction with dithionite, the ν 4 oxidation-state marker band shifts to 1360 cm −1 while the ν 3 and ν 10 at 1471 and 1604 cm −1 , respectively, are indicative of a 5-coordinate high-spin configuration (Figure 7). Binding of CO up-shifts these frequencies (ν 4 at 1374, ν 3 at 1503, and ν 10 at 1638 cm −1 ) and results in the detection of a ν (Fe-CO) at 533 cm −1 (Figure 8), which is characteristic of heme-carbonyl complexes with a weak or no proximal ligand (24,(26)(27)(28). Figure 9 shows the electrochemistry of wild-type Dap1p as evaluated using UV-Vis spectroelectrochemistry.…”