Properties of Hb Wood (beta-97(FG4)His leads to Leu), a high oxygen affinity hemoglobin with reduced hemeheme interaction, were examined in its nitric oxide liganded form. The reactivity of the beta-93 thiol groups and the electron paramagnetic resonance (EPR) spectrum were examined to determine what effect the amino acid substitution, which occurs at the alpha1beta2 interface, would have on inositol hexaphosphate induced transition of this form of the tetramer. Binding of inositol hexaphosphate (IHP) in a 1:1 stoichiometry was demonstrated. In spite of apparently normal interaction with IHP, there was little or no change in the reactivity of the beta-93 thiol groups and in the electron paramagnetic resonance (EPR) spectrum as contrasted with the marked changes characteristic of normal hemoglobin (HbA). In contrast with NO-HbA, there was also no development of the EPR hyperfine structure in NO-Hb Wood with increased protonation of the protein at pH below 7.0. Taken together with the observations of Henry and Banerjee ((1973), J. Mol. Biol. 73, 469) on the development of NO-Hb EPR hyperfine structure and of Perutz et al. (1974a), Biochemistry 13, 2174) on changes in thiol reactivity with the R leads to T transition, the results suggest that IHP or H+ cannot switch NO-Hb Wood to the T conformation. Since the atomic structures of met- and deoxyhemoglobin offer no indication that His-97 plays any special part in the allosteric mechanism (M. E. Perutz, personal communication), it appears that the replacement of His-97 by Leu reduces the stability of the T structure relative to that of R.

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prepared 8s an account of work sponsored by the United States Oovernment. Neither the United States nor the United Statw Atomic &nergy Conmidon, nor my of their employ-, nor m y of weir contracton, suboontsactors, or thefr employ-, make8 any m m n t y , e X p m UI implird, or as8uma my legal liability or responribflfty for the accurrcy, aomp l e t e n~ or usefulneo of any infomation, apparatus, p d u o t . o r process disclosed, or repremnts that its use would not infrhge privately owned xi%@. I DISCLAIMERThis report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency Thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. DISCLAIMERPortions of this document may be illegible in electronic image products. Images are produced from the best available original document.This report was prepared as an account of work sponsored by the United States Government. Neither the United States nor the United States Atomic Energy Commission, nor any of their employees, nor any of their c'ontractors, subcontractors, or their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness or usefulness of any information, apparatus, product or process disclosed, or represents that its use would not infringe privately owned rights. EXPERIMENTAL I n t r o d u c t i o n EPR s p e c t r o m e t e r Dewar and sample arrangements V i s i b l e and i n f r a r e d equipment C r y s t a l s EPR of NbBrqAc2Powder C r y s t a l s Glasses F u t u r e work E l e c t r o n i c S p e c t r a of NbBrqAc2 Experimental g-values were compared with theoretical g-value expressions. From this comparison, the first excited s t a t e was estimated t o be approximately 2100 cm" above t h e ground s t a t e and t h e f o u r t h e x c i t e d s t a t e was estimated t o be approximately 25,500 cm-' above t h e ground s t a t e . The d i f f e re n c e i n t h e experimental and t h e c a l c u l a t e d values of gZz i n d i c a t e d t h a t t h e o c t a h e d r a l c r y s t a l f i e l d wave f u n c t i o n s d i d not e x p l a i n t h e .EPR. d a t a . A f t e r r e d u c t i o n t o t h e C2v p o i n t 'symmetry t h e e x c i t e d s t a t e s with an A 1 r e p r e s e n t a t i o n can mix.Also, some mixing of ligand o r b i t a l s with m...

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