The Role of Electronic Excitation States in Collagen Biosynthesis

THE ROLE OF ELECTRONIC EXCITATION STATES IN COLLAGEN BIOSYNTHESIS* RANDOLPH M. HOWES,f RICHARD H. STEELE, t andJOHN E. HOOPESf Nature's ability to heal wounds in spite of balms, powders, lotions, salves, and ointments demonstrates her power to overcome adversity. In attempts to discover their mechanistic secrets, wounds have been stressed, stretched, torn, minced, stained, assayed, and treated with a wide variety oftopical agents and dressings [I]. Yet man has gained little control, if any, over healing and scarring rates [2]. A review of the literature reveals a striking similarity between the reaction mechanisms of the microsomal mixed-function oxygenases and proline hydroxylation systems. These data suggest that both systems probably involve electronic excitation states. Proline Hydroxylation The data indicating that the hydroxylation of peptidyl proline in peptide chains is the rate-limiting step in collagen synthesis are presently inconclusive. Nonetheless, this reaction appears to be the most specific reaction in the biochemical pathway ofcollagen synthesis, as is evidenced in studies with mammalian tissues which show that prolyl hydroxylase activity reflects the rate ofcollagen synthesis [3-8]. Investigations on the biosynthesis ofcollagen and formation of hydroxyproline have revealed a requirement for atmospheric oxygen [9, 10], iron [11-14], and alphaketoglutarate [15]. Information by Fujimoto and Tamiya [9] concerning the mechanism of hydroxylation has shown that the hydroxyl moiety of collagen hydroxyproline is derived from molecular oxygen and not from water, ?This work was supported in part by a grant from the Southern Medical Association and the Educational Foundation of the American Society of Plastic and Reconstructive Surgery. tDivision of Plastic Surgery, The Johns Hopkins University School of Medicine, Baltimore , Maryland 21205. ^Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112. Perspectives in Biology and Medicine ¦ Summer 1977 | 539 and data by Fujita et al. [16] verified the hypothesis that hydroxyproline formation is catalyzed by an oxygenase system. Studies by Prockop, Kaplan, and Udenfriend [10]; by Lowther, Green, and Chapman [17]; and by Eastoe [18] demonstrated (a) that collagen hydroxyproline-C14 first appeared in tissue incubations in the microsomes, (¿>) that synthesis ofcollagen is a microsomal process, and (c) that interpretations indicated that proline hydroxylation was also catalyzed by an enzymatic oxygenase or mixed-function oxidase system. The Oxygenases The oxygenase microsomal enzymatic system is responsible for the metabolism of drugs, insecticides, xenobiotics, and carcinogenic compounds ; likewise it is concerned with the transformation of various essential metabolites which include hydroxylation reactions such as detoxification mechanisms; the breakdown of aromatic and cyclic compounds into small fragments; and the transformation, degradation, and synthesis of various steroid hormones. These reactions require an oxidizable substrate, a reduced pyridine nucleotide which is usually NADPH, and molecular oxygen [19]. One of the major unsolved features of the mixed-function oxidations concerned the specific nature of the "active" oxygen species which effected the oxidation of the substrates . Considerations of the literature led Ullrich and Staudinger [20] to reject the hydroxyl radical (-OH), the superoxide radical (-???) and its anion (·00> the hydroxyl radical cation (OH+), hydrogen peroxide (HOOH), ground-state oxygen (3Oi), the oxenoid species (O2—, the completely dissociated, conjugate base of HOOH, as a metallic complex, e.g., Fe2+00), and the oxene atom (:0:) as being those "active" oxygen species responsible for microsomal oxidations. Relationship ofHydroxylation to Peroxidation and Electronic Excitation States Orrenius, Dallner and Ernster [21] and Gotto et al. [22], found that drugs undergoing NADPH-linked hydroxylation in liver microsomes inhibited lipid peroxidation. Wills [23] considered the possibility that the two processes of hydroxylation and lipid peroxidation were closely linked in microsomes and dependent upon the same electron transport chain. Howes and Steele [24] described a chemiluminescence evoked from rat-liver microsomes by NADPH and oxygen in the absence of an oxidizable substrate. Howes and Steele [25] subsequently showed that chemiluminescence and lipid peroxidation were suppressed with the initiation of hydroxylation upon the addition of appropirate substrates 540 I Randolph M. Howes, Richard H. Steele, andJohn E. Hoopes ¦ Collagen Biosynthesis to the system. These works indicated that NADPH-induced microsomal chemiluminescence, lipid peroxidation, and substrate hydroxylations were mutually competitive processes. They further proposed that these processes were mediated by electronically excited singlet molecular oxygen with the generation and utilization ofelectronic excitation states...