Abstract:
:Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding. The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through site-directed mutagenesis. Substitution of His-E7 with glycine [GlyE7]Mb bound with CO (CO[GlyE7]Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1. After flash photolysis at 10 K, the unbound state of CO[GlyE7]Mb exhibits two CO stretch bands, whereas MbCO has three. Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to [GlyE7]Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal. Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for [GlyE7]MbCO as compared with wild-type MbCO. These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure.
journal_name
Proc Natl Acad Sci U S Aauthors
Braunstein D,Ansari A,Berendzen J,Cowen BR,Egeberg KD,Frauenfelder H,Hong MK,Ormos P,Sauke TB,Scholl Rdoi
10.1073/pnas.85.22.8497subject
Has Abstract,Author List Incompletepub_date
1988-11-01 00:00:00pages
8497-501issue
22eissn
0027-8424issn
1091-6490journal_volume
85pub_type
杂志文章abstract::The chloroplast stromal proteins from spinach condense the two heterocyclic moieties of vitamin B1--4-methyl-5-(beta-hydroxyethyl)thiazole and 2-methyl-4-amino-5-hydroxymethylpyrimidine--in the presence of MgATP. We have taken advantage of this observation to study thiazole synthesis. We present data indicating that p...
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