Solution 1H NMR of the active site of substrate-bound, cyanide-inhibited human heme oxygenase. comparison to the crystal structure of the water-ligated form.
ABSTRACT The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer ( approximately 75-80%) in solution is rotated 180 degrees about the alpha-gamma-meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 A closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha-meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a approximately 20 degrees tilt of the distal ligand from the heme normal in the direction of the alpha-meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta, gamma, and delta-meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha-meso position.
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ABSTRACT: The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has been definitively observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881-1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252-2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75% and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate kBA = (3.6 ps)-1 is the dominant process, some CN- exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN- association rate, we find that the CN- escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ~ 1-2 x107 s-1. This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1x107 s-1) under the same conditions. The analysis leads to an escape probability kout/(kout+kBA) ~ 10-4, which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN- bimolecular association rate (170 M-1s-1), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN- bimolecular association rate is larger by ~103 making the CN- photolysis more difficult to observe.The Journal of Physical Chemistry B 03/2013; 117(15). DOI:10.1021/jp401224f · 3.38 Impact Factor
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ABSTRACT: A correspondence between networks and gels, physical or chemical, and native globular proteins is established by visualizing proteins as physical gels. The emphasis is placed on spatial fluctuations of residues in proteins which are correlated, and it is stated that these correlations determine the function of the proteins. Structural and topological features necessary for a gel to exhibit features of a protein such as to perform a predetermined function are discussed. A simple mathematical model that explains the relationships between correlated fluctuations of residues and the function of the protein is given.Macromolecular Symposia 09/2011; 306-307(1). DOI:10.1002/masy.201000118
- Biochemistry 09/2001; 40(38):11552-11558. DOI:10.1021/bi0110239 · 3.19 Impact Factor