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Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.

A biochemical molecular modeling project on heme proteins suitable for an introductory Biochemistry I class has been designed with a 2-fold objective: i) to reinforce the correlation between protein threedimensional structure and function through a discovery oriented project, and ii) to introduce students to the fields of bioinorganic and coordination chemistry. Students are asked to identify several unknown heme proteins based on a careful analysis of covalent and noncovalent interactions at the active site of each protein, focusing on amino acid reactivity and H-bonding networks. Starting with the three-dimensional crystal structures of four unknown proteins, students isolate and examine the coordination environment of the iron center in order to predict the relative reactivity toward dioxygen (O 2 ) or hydrogen peroxide (H 2 O 2 ). The central question of the project is to determine how the same iron protoporphyrin IX cofactor can be used by four different proteins to carry out diverse reactions, from electron transfer, to reversible oxygen binding to hydrogen peroxide activation. Pedagogical reasons for implementation of this biomolecular discovery-based activity and student evaluations are discussed. In addition to developing many of the three-dimensional visualization skills needed to successfully learn biochemistry, students also learn to use the versatile MOE molecular modeling program (Molecular Operating Environment), become familiar with metalloprotein reactivity, and are introduced to computational biochemistry research.